AU2016273912A1 - High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris - Google Patents

Abstract

Methods for producing heterologous multi-subunit proteins in transformed cells are disclosed. In particular, the present disclosure provides improved methods of producing multi-subunit proteins, including antibodies and other multi-subunit proteins, which may or may not be secreted, with a higher yield and decreased production of undesired side-products. In exemplary embodiments, the transformed cells are a yeast, e.g., methylotrophic yeast such as Pichia pastoris.

Description

2016273912 14 Dec 2016

HIGH-PURITY PRODUCTION OF MULTI-SUBUNIT PROTEINS SUCH AS ANTIBODIES IN TRANSFORMED MICROBES SUCH AS PICHIA PASTORIS

RELATED APPLICATION DISCLOSURE

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 61/525,307 (Atty. Docket No. 67858.730200), filed August 19, 2011 (entitled “MULTI-COPY STRATEGY FOR HIGH-TITER AND HIGH-PURITY PRODUCTION OF MULTI-SUBUNIT PROTEINS SUCH AS ANTIBODIES IN TRANSFORMED MICROBES SUCH AS PICHIA PASTORIS,” U.S. Provisional Application Ser. No. 61/488,660 (Atty. Docket No. 67858.730300), filed May 20, 2011, entitled “ANTI-CGRP COMPOSITIONS AND USE THEREOF,” U.S. Provisional Application Ser. No. 61/496,860 (Atty. Docket No. 67858.760000), filed June 14, 2011, entitled “USE OF ANTI-CGRP ANTIBODIES AND ANTIBODY FRAGMENTS TO PREVENT OR INHIBIT PHOTOPHOBIA IN SUBJECTS IN NEED THEREOF, ESPECIALLY MIGRAINE SUFFERERS,” and U.S. Provisional Application Ser. No. 61/496,873 (Atty. Docket No. 67858.770000), filed June 14, 2011, entitled “USE OF ANTI-CGRP ANTIBODIES AND ANTIBODY FRAGMENTS TO TREAT DIARRHEA IN SUBJECTS WITH DISEASES OR TREATMENTS THAT RESULT IN ELEVATED CGRP LEVELS” each of which is incorporated by reference herein in its entirety.

[0002] This application includes a Sequence Listing which is being submitted in ASCII format via EFS-Web, in a file named “67858o711002.txt” created May 8, 2012 and having a size of 315,392 bytes, which is hereby incorporated by reference in its entirety.

FIELD

[0003] The present disclosure generally relates to methods for producing heterologous proteins in transformed cells. In particular, the present disclosure provides improved methods of producing multi-subunit proteins, including antibodies and other multi-subunit proteins, which may or may not be secreted, with decreased production of undesired side-products and/or increased yield. In exemplary embodiments, the transformed cells are a yeast, such as Pichia pastoris or Saccharomyces cerevisiae. 1 2016273912 14 Dec 2016

BACKGROUND

[0004] Conventional antibodies are tetrameric proteins composed of two identical light chains and two identical heavy chains. Pure human antibodies of a specific type can be difficult or impossible to purify from natural sources in sufficient amounts for many purposes. As a consequence, biotechnology and pharmaceutical companies have turned to recombinant DNA-based methods to prepare antibodies on a large scale. The production of functional antibodies generally involves not just the synthesis of the two polypeptides but also a number of post-translational events, including proteolytic processing of the N-terminal secretion signal sequence; proper folding and assembly of the polypeptides into tetramers; formation of disulfide bonds; and typically includes a specific N-linked glycosylation. All of these events take place in the eukaryotic cell secretory pathway, an organelle complex unique to eukaryotic cells.

[0005] Recombinant synthesis of such complex proteins has typically relied on cultures of higher eukaryotic cells to produce biologically active material, with cultured mammalian cells being very commonly used. However, mammalian tissue culture-based production systems incur significant added expense and complication relative to microbial fermentation methods. Additionally, products derived from mammalian cell culture may require additional safety testing to ensure freedom from mammalian pathogens (including viruses) that might be present in the cultured cells or animal-derived products used in culture, such as serum.

[0006] Prior work has help to establish the yeast Pichia pastoris as a cost-effective platform for producing functional antibodies that are potentially suitable for research, diagnostic, and therapeutic use. See co-owned U.S. Patents 7,935,340 and 7,927,863, each of which is incorporated by reference herein in its entirety. Methods are also known in the literature for design and optimization of P. pastoris fermentations for expression of recombinant proteins, including optimization of the cell density, broth volume, substrate feed rate, and the length of each phase of the reaction. See Zhang et al., “Rational Design and Optimization of Fed-Batch and Continuous Fermentations” in Cregg, J. M., Ed., 2007, Pichia Protocols (2nd edition), Methods in Molecular Biology, vol. 389, Humana Press, Totowa, N.J., pgs. 43-63.

[0007] Though recombinant multi-subunit proteins can be produced from cultured cells, undesired side-products may also be produced. For example, the cultured cells may produce the 2 2016273912 14 Dec 2016 desired multi-subunit protein along with free monomers, complexes having incorrect stoichiometry, or proteins having undesired or aberrant glycosylation. Purification of the desired multi-subunit protein can increase production cost, and the steps involved in purification may decrease total yield of active complexes. Moreover, even after purification, undesired side-products may be present in amounts that cause concern. For example, glycosylated side-products may be present in amounts that increase the risk of an immune reaction after administration, while aberrant complexes or aggregates may decrease specific activity and may also be potentially immunogenic.

SUMMARY

[000S] Most IgGl antibody molecules are stabilized by a total of 16 intra-chain and interchain disulfide bridges. The intra-chain disulfide bridges stabilize the folding of the IgG domains in both heavy and light chains, while the inter-chain disulfide bridges stabilize the association between heavy and light chains. As a result of these bonds, antibodies form a stable complex containing two heavy chains and two light chains (H2L2). However, due to improper disulfide bond formation, product-associated variants are sometimes found in recombinant antibody preparations, including a complex having one light and one heavy chain (HILI) and a complex having two heavy chains and one light chain (H2L1). Further, higher order complexes may also form in which additional inter-chain disulfide bonds form, resulting in a greater number of covalently linked subunits.

[0009] As further described below, Applicants have now identified methods of decreasing the production of these complexes containing aberrant disulfide bonds during recombinant production of antibodies from yeast culture. Specifically, the method involves addition of an ethanol bolus to the culture, and resulted in decreased production of the H1L1, H2L1, and H4L4 products-associated variants, and increased purity of the desired H2L2 product. The H1L1 and H2L1 complexes were detected by non-reduced, denaturing SDS-PAGE, and the H4L4 complexes were detected by size exclusion chromatography. Using the subject methods, proper disulfide bond formation was facilitated, resulting in increased antibody purity. This was demonstrated for three different antibodies, all three of which exhibited improved purity when produced with the addition of the ethanol bolus (FIGS. 1-6). These three antibodies are not only are different in sequence but also recognize three different antigens. Moreover, when produced 3 2016273912 14 Dec 2016 in the absence of an ethanol bolus, two of the antibodies contained greater amounts of the H1L1 product (FIGS. 1-4), compared with the third antibody (FIG. 5). The two antibodies containing greater amounts of the H1L1 product have a non-canonical or additional disulfide bridge, whereas the third does not. The antibody exemplified in FIGS. 1, 2 and 3 has an additional intrachain disulfide bridge in the variable light chain domain, while the antibody exemplified in FIG. 4, has an additional intra-chain disulfide bridge in its heavy chain. It has been reported in the literature that the presence of disulfide-bridges in overexpressed proteins increases intracellular stress in the host (see Gasser et al., Biotechnology and Bioengineering, Vol. 94, No. 2, pg. 353-61, June 5, 2006; Inan et al., Biotechnology And Bioengineering, Vol. 93, No. 4, pg. 771-78, March 5, 2006; Li et al., Biochem Biophys Res Commun. 2010 November 19; 402(3): 519-524). This increased stress can also lead to lower viability, as is demonstrated in FIGS. 11, 12, and 13 where both antibodies with the extra intra-chain disulfide bridge have lower viability under the “no-bolus” conditions. The addition of the ethanol bolus, therefore leads to increased viability and increased purity. This may be of use in particular when difficult to express proteins with multiple disulfide bridges are being expressed.

[0010] In one aspect, the present disclosure provides a method of producing a multisubunit complex, comprising: (a) providing a culture comprising a eukaryotic cells comprising genes that provide for the expression of the subunits of said multi-subunit complex; (b) adding a bolus of ethanol to said culture; and (c) culturing said culture to produce said multi-subunit complex.

[0011] The ethanol bolus may enhance the formation of stable disulfide bonds relative to the same method effected in the absence of the bolus of ethanol.

[0012] Said multi-subunit complex may contain one or more polypeptides comprising at least one disulfide bond.

[0013] Said multi-subunit complex may comprise an antibody.

[0014] The method may decrease the relative abundance of one or more product-associated variants relative to the same method effected in the absence of the bolus of ethanol. 4 2016273912 14 Dec 2016 [0015] The method may decrease the relative abundance of product-associated variants having a higher or lower apparent molecular weight than said desired multi-subunit complex as detected by size exclusion chromatography or gel electrophoresis relative to the same method effected in the absence of the bolus of ethanol.

[0016] The method may decrease the relative abundance of complexes having aberrant stoichiometry relative to the same method effected in the absence of the bolus of ethanol.

[0017] The method may decrease the relative abundance of complexes having aberrant disulfide bonds relative to the same method effected in the absence of the bolus of ethanol.

[0018] The method may decrease the relative abundance of complexes having reduced cysteines relative to the same method effected in the absence of the bolus of ethanol.

[0019] The method may decrease the relative abundance of complexes having aberrant glycosylation relative to the same method effected in the absence of the bolus of ethanol.

[0020] The method may modulate the formation or stability of inter-heavy chain disulfide bonds.

[0021] The method may modulate the formation or stability of disulfide bonds linking the light and heavy chains.

[0022] The method may decrease the relative abundance of one or more product-associated variants relative to the same method effected in the absence of the bolus of ethanol.

[0023] Said product-associated variants may comprise one or more of the Η1L1, H2L1, and H4L4 product-associate variants.

[0024] The method increase the purity of said antibody relative to said method effected in the absence of said bolus of ethanol.

[0025] Step (b) may be effected prior to step (c).

[0026] Step (b) may be effected subsequent to step (c). 5 2016273912 14 Dec 2016 [0027] Step (b) may be effected concurrently with step (c).

[0028] Step (b) may result in a concentration of ethanol in said culture of between about 0.01% and about 4% (w/v).

[0029] Step (b) may result in a concentration of ethanol in said culture of between about 0.01% and about 4%, between about 0.02% and about 3.75%, between about 0.04% and about 3.5%, between about 0.08% and about 3.25%, between about 0.1% and about 3%, between about 0.2% and about 2.75%, between about 0.3% and about 2.5%, between about 0.4% and about 2.25%, between about 0.5% and about 1.5%, between about 0.5% and about 2%, between about 0.6% and about 1.75%, between about 0.7% and about 1.5%, or between about 0.8% and about 1.25%.

[0030] Step (b) may result in a concentration of ethanol in said culture that may be at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.6%, 0.6%, 0.7%, 0.8% or 0.9% (w/v).

[0031] Step (b) may result in a concentration of ethanol in said culture that may be at most about 4%, 3.5%, 3%, 2.5%, 2%, 1.8%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, or 0.15% (w/v).

[0032] Step (b) may comprise adding ethanol to said culture, adding a carrier comprising ethanol to said culture, adding said cells to a medium or carrier comprising ethanol, or replacing part of the culture medium.

[0033] Said bolus of ethanol may be added to the culture medium over a period of time between 1 and 20 minutes.

[0034] Step (c) may comprise providing oxygen to said cells.

[0035] Said providing oxygen may comprise agitating said culture.

[0036] Said providing oxygen may comprise contacting said culture with a gas mixture comprising oxygen. 6 2016273912 14 Dec 2016 [0037] Step (c) may comprise adding a feed comprising a carbon source to said culture.

[0038] Said feed may comprise at least one fermentable carbon source.

[0039] Said feed may comprise one or more of glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose, mannitol, and raffmose.

[0040] The method may further comprise maintaining the concentration of ethanol between an upper set point and a lower set point during step (c).

[0041] Said lower set point may be about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.6%, 0.6%, 0.7%, 0.8% or 0.9% (w/v).

[0042] Said upper set point may be about 4%, 3.5%, 3%, 2.5%, 2%, 1.8%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, or 0.15% (w/v).

[0043] Said upper set point may be at most about 1.5%, 1.4%, 1.3, 1.2%, or 1.1% (w/v).

[0044] The method may further comprise maintaining the concentration of ethanol at a set point during step (c).

[0045] Said set point may be about 0.1%, 0.2%, 0. 3%, 0. 4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 01.%, 01.1%, 01.2%, 01.3%, 01.4%, or 01.5% (w/v).

[0046] Step (c) may comprise maintaining the concentration of ethanol in said culture between about 0.01% and about 4%, between about 0.02% and about 3.75%, between about 0.04% and about 3.5%, between about 0.08% and about 3.25%, between about 0.1% and about 3%, between about 0.2% and about 2.75%, between about 0.3% and about 2.5%, between about 0.4% and about 2.25%, between about 0.5% and about 2%, between about 0.6% and about 1.75%, between about 0.7% and about 1.5%, or between about 0.8% and about 1.25%.

[0047] The concentration of ethanol in said culture may be maintained by controlling production of ethanol by said cells or by addition of ethanol to said culture. 7 2016273912 14 Dec 2016 [0048] The step of controlling production of ethanol may comprise controlling one or more of the concentration of glucose, availability of oxygen, intensity of agitation, gas pressure, flow rate of supplied air or other gas mixture, viscosity of the culture, culture density, concentration of oxygen in the supplied air or other gas mixture, and temperature.

[0049] The time between step (a) and step (b) may be less than about 72 hours, less than about 48 hours, less than about 24 hours, less than about 12 hours, less than about 9 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 90 minutes, less than about 30 minutes, less than about 5 minutes, or less than about 1 minute.

[0050] The time between step (b) and step (c) may be less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, less than about 90 minutes, less than about 80 minutes, less than about 70 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 30 minutes, less than about 20 minutes, less than about 10 minutes, less than about 5 minutes, or less than about 1 minute.

[0051] The culture of step (a) may be produced by adding a carbon source to said culture, and culturing said culture until the carbon source may be depleted.

[0052] Said carbon source may comprise one or more of: glycerol, glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose, mannitol, and raffmose.

[0053] The depletion of the carbon source may be determined by detecting a decrease in the metabolic activity of said eukaryotic cells.

[0054] Said decrease in the metabolic activity of said eukaryotic cells may be identified by detecting a decrease in the consumption of oxygen by said eukaryotic cells, by detecting an increase in pH in the culture, by detecting stabilization of the wet cell mass, or by detecting an increase in the concentration of ammonia in the culture. 8 2016273912 14 Dec 2016 [0055] Said decrease in the consumption of oxygen by said eukaryotic cells may be identified by detecting an increase in the concentration of dissolved oxygen in said culture.

[0056] Said eukaryotic cells may comprise yeast cells.

[0057] Said yeast cells may comprise methylotrophic yeast.

[0058] Said methylotrophic yeast may be of the genus Pichia.

[0059] Said methylotrophic yeast of the genus Pichia may be Pichia pastoris.

[0060] Said methylotrophic yeast of the genus Pichia may be selected from the group consisting of: Pichia angusta, Pichia guillermordii, Pichia methanolica, and Pichia inositovera.

[0061] The genes that provide for expression of said multi-subunit complex may be integrated into one or more genomic loci.

[0062] At least one of said genomic loci may be selected from the group consisting of the pGAP locus, 3’ AOX TT locus; PpURA5; OCH1; AOX1; HIS4; GAP; pGAP; 3’ AOX TT; ARG; and the HIS4 TT locus.

[0063] At least one of the genes encoding said subunits of the multi-subunit complex may be expressed under control of an inducible or constitutive promoter.

[0064] Said inducible promoter may be selected from the group consisting of the AOX1, CUP1, tetracycline inducible, thiamine inducible, and FLD1 promoters.

[0065] At least one of the genes encoding said subunits of the multi-subunit complex may be expressed under control of a promoter selected from the group consisting of: the CUP1, AOX1, ICL1, glyceraldehyde-3-phosphate dehydrogenase (GAP), FLD1, ADH1, alcohol dehydrogenase II, GAL4, PH03, PH05, and Pyk promoters, tetracycline inducible promoters, thiamine inducible promoters, chimeric promoters derived therefrom, yeast promoters, mammalian promoters, insect promoters, plant promoters, reptile promoters, amphibian promoters, viral promoters, and avian promoters.

[0066] Said eukaryotic cell may be a diploid, tetraploid cell, or polyploid. 9 2016273912 14 Dec 2016 [0067] The method may further comprise purifying said multi-subunit complex from said eukaryotic cells or from the culture medium.

[0068] Said multi-subunit complex may be purified from an intracellular component, cytoplasm, nucleoplasm, or a membrane of said eukaryotic cells.

[0069] Said eukaryotic cells secrete said multi-subunit complex into the culture medium. [0070] Said multi-subunit complex may be purified from said culture medium. [0071] Said multi-subunit complex may comprise a monospecific or bispecific antibody.

[0072] Said multi-subunit complex may comprise a human antibody or a humanized antibody or fragment thereof.

[0073] Said humanized antibody may be of mouse, rat, rabbit, goat, sheep, or cow origin. [0074] Said humanized antibody may be of rabbit origin. [0075] antibody. Said multi-subunit complex may comprise a monovalent, bivalent, or multivalent [0076] affinity. Said antibody may be purified from said culture by protein A and/or protein G [0077] At least one of the genes that provide for expression of a subunit of said multi- subunit complex in at least one of said eukaryotic cells in said panel may be optimized for expression in said eukaryotic cell.

[0078] Said multi-subunit complex may comprise an antibody and the purity of said antibody may be assessed by measuring the fraction of the antibody produced by said eukaryotic cell that may be contained in antibody complexes having the expected apparent hydrodynamic radius, may be contained in antibody complexes having the expected molecular weight, and / or specifically binds a target of said antibody. 10 2016273912 14 Dec 2016 [0079] Said multi-subunit complex may comprise an antibody and the yield of said antibody may be assessed by determining the amount of antibody produced by said eukaryotic cell discounting any product-associated variants that may be abnormally glycosylated, contained in antibody complexes other than complexes having the expected apparent hydrodynamic radius, contained in antibody complexes having the expected molecular weight, and / or that fail to specifically bind to the target of said antibody.

[0080] The molecular weight of said antibody complexes may be determined by nonreducing SDS-PAGE.

[0081] Said multi-subunit complex may comprise an antibody, said method may further comprise purifying said antibody.

[0082] Said culture cell may produce a supernatant antibody titer of at least 100 mg / L, at least 150 mg / L, at least 200 mg / L, at least 250 mg / L, at least 300 mg / L, between 100 and 300 mg / L, between 100 and 500 mg / L, between 100 and 1000 mg / L, at least 1000 mg / L, at least 1250 mg/liter, at least 1500 mg/liter, at least about 1750 mg/liter, at least about 2000 mg/liter, at least about 10000 mg/liter, or more.

[0083] One or more subunits of said multi-subunit complex may be expressed from more than one gene copy.

[0084] Said multi-subunit complex may comprise an antibody which may be expressed from between 1-10 copies of a gene encoding the light chain of said antibody and from 1-10 copies of a gene encoding the heavy chain of said antibody.

[0085] The genes that provide for expression of said multi-subunit complex may be integrated into genome of said cells.

[0086] The genes that provide for expression of said multi-subunit complex may be contained on an extrachromosomal element, plasmid, or artificial chromosome.

[0087] Said cells may comprise more copies of the gene that provide for the expression of the light chain of said antibody than copies of the gene that provide for expression of the heavy chain of said antibody. 11 2016273912 14 Dec 2016 [0088] The respective number of copies of the gene encoding the heavy chain of said antibody and the number of copies of the gene encoding the light chain of said antibody in said cells may be: 2 and 2, 2 and 3, 3 and 3, 3 and 4, 3 and 5, 4 and 3, 4 and 4, 4 and 5, 4 and 6, 5 and 4, 5 and 5, 5 and 6, or 5 and 7.

[0089] The respective number of copies of the gene encoding the heavy chain of said antibody and the number of copies of the gene encoding the light chain of said antibody in said cells may be: 2 and 1, 3 and 1, 4 and 1, 5 and 1, 6 and 1, 7 and 1, 8 and 1, 9 and 1, 10 and 1, 1 and 2, 2 and 2, 3 and 2, 4 and 2, 5 and 2, 6 and 2, 7 and 2, 8 and 2, 9 and 2, 10 and 2, 1 and 3, 2 and 3, 3 and 3, 4 and 3, 5 and 3, 6 and 3, 7 and 3, 8 and 3, 9 and 3, 10 and 3, 1 and 4, 2 and 4, 3 and 4, 4 and 4, 5 and 4, 6 and 4, 7 and 4, 8 and 4, 9 and 4, 10 and 4, 1 and 5, 2 and 5, 3 and 5, 4 and 5, 5 and 5, 6 and 5, 7 and 5, 8 and 5, 9 and 5, 10 and 5, 1 and 6, 2 and 6, 3 and 6, 4 and 6, 5 and 6, 6 and 6, 7 and 6, 8 and 6, 9 and 6, 10 and 6, 1 and 7, 2 and 7, 3 and 7, 4 and 7, 5 and 7, 6 and 7, 7 and 7, 8 and 7, 9 and 7, 10 and 7, 1 and 8, 2 and 8, 3 and 8, 4 and 8, 5 and 8, 6 and 8, 7 and 8, 8 and 8, 9 and 8, 10 and 8, 1 and 9, 2 and 9, 3 and 9, 4 and 9, 5 and 9, 6 and 9, 7 and 9, 8 and 9, 9 and 9, 10 and 9, 1 and 10, 2 and 10, 3 and 10, 4 and 10, 5 and 10, 6 and 10, 7 and 10, 8 and 10, 9 and 10, 10 and 10.

[0090] The culture of step (c) may be grown in a production medium.

[0091] Said production medium may be a minimal medium.

[0092] Said minimal medium lacks selective agents.

[0093] Said minimal medium lacks pre-formed amino acids or other complex biomolecules.

[0094] The production medium may be a complex medium.

[0095] The complex medium may comprise one or more of yeast extract, soy peptones, and other plant peptones.

[0096] The culture of step (c) may be grown to a high cell density.

[0097] Said high cell density may be at least 50 g/L. 12 2016273912 14 Dec 2016 [0098] Said high cell density may be at least 100 g/L.

[0099] Said high cell density may be at least 300 g/L.

[00100] Said high cell density may be at least 400 g/L.

[00101] Said high cell density may be at least 500 g/L.

[00102] Said high cell density may be at least 750 g/L.

[00103] The yeast cells may be cultured for at least 20 doublings and maintain high levels of expression of said multi-subunit complex after said at least 20 doublings.

[00104] The cells of step (c) may be cultured for at least 50 doublings and maintain high levels of expression of said multi-subunit complex after said at least 50 doublings.

[00105] The cells of step (c) may be cultured for at least 100 doublings and maintain high levels of expression of said multi-subunit complex after said at least 100 doublings.

[00106] At least one subunit of said multi-subunit complex may comprise a secretion signal.

[00107] Said multi-subunit complex may comprise an antibody.

[00108] The secretion signal may comprise one or more polypeptides selected from the group consisting of: SEQ ID NOS: 414 to 437 and any combination thereof.

[00109] Said multi-subunit complex may be not any of the antibodies disclosed in U.S. Provisional Application No. 61/418,832, filed December 1, 2010, PCT/US11/62963, filed December 1, 2011, U.S. Ser. No. 13/309,295, filed December 1, 2011, U.S. Ser. No. 13/309,153, filed December 1, 2011, U.S. Ser. No. 13/308,665 filed on December 1, 2011, and U.S. Ser. No. 13/308,831, filed December 1, 2011.

[00110] Said multi-subunit complex may not be Abl-NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, AblO-NGF, Abl 1-NGF, Abl2-13 2016273912 14 Dec 2016 NGF, AM3-NGF, Abl4-NGF, AM5-NGF, Abl6-NGF, Abl7-NGF, Abl8-NGF, Abl9-NGF, Ab20-NGF, and Ab21-NGF, or an Fab2 or Fabl fragment thereof.

[00111] Said multi-subunit complex may not contain at least one, at least two, at least three, at least four, at least five, or at least all six of the complementarity determining regions (CDRs) contained in any of the following antibodies: Abl-NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, AblO-NGF, Abll-NGF, Abl2-NGF, Abl3-NGF, AM4-NGF, Abl5-NGF, Abl6-NGF, Abl7-NGF, Abl8-NGF, Abl9-NGF, Ab20-NGF, or Ab21-NGF and optionally having binding specificity for NGF.

[00112] Said multi-subunit complex may not comprise or consist of the light and heavy chain polypeptide sequences of SEQ ID NOs: 51 and 401, respectively, SEQ ID NOs: 53 and 402, respectively, SEQ ID NOs: 405 and 406, respectively, and SEQ ID NOs: 407 and 408, respectively.

[00113] Said multi-subunit complex may not comprise an antibody containing at least one, at least two, at least three, at least four, at least five, or at least all six of the CDRs of SEQ ID NOs: 55, 56, 57, 58, 59, and 60, and optionally having binding specificity for NGF.

[00114] Said multi-subunit complex may not comprise any of the antibodies or antibody coding sequences disclosed herein in the sections entitled “Anti-NGF Antibodies and Binding Fragments Thereof Having Binding Activity for NGF” and “Polynucleotides Encoding Anti-NGF Antibody Polypeptides.”In one aspect, the present disclosure provides methods of producing a multi-subunit complex, which may comprise: culturing a host cell providing a culture comprising eukaryotic cells that expresses said multi-subunit complex, adding a bolus 7of ethanol to said culture, and culturing said culture to produce said multi-subunit complex.

The multi-subunit complex may comprise one or more disulfide bonds, and may be an antibody.

[00115] The ethanol bolus concentration (expressed as % w/v) may be between about 0.1% and about 5%, such as at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, up to about 1%, up to about 1.1%, up to about 1.2%, up to about 1.3%, up to about 1.4%, up to about 1.5%, up to about 1.6%, up to about 1.7%, up to about 14 2016273912 14 Dec 2016 1.8%, up to about 1.9%, up to about 2%, up to about 3%, up to about 4%, or up to about 5%, such as between about 0.1% and about 1.9%, between about 0.2% and about 1.8%, between about 0.3% and about 1.7%, between about 0.4% and about 1.6%, between about 0.5% and about 1.5%, between about 0.6% and about 1.4%, between about 0.7% and about 1.3%, between about 0.8% and about 1.2%, or between about 0.9% and about 1.1%, such as about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.5%, about 3%, about 4%, or about 5%.

[00116] The method may further comprise purification of said desired multi-subunit complex.

[00117] In exemplary embodiments the ethanol concentration may be controlled subsequent to addition of the ethanol bolus, which may be used to maintain the ethanol concentration at a desired set point or within a desired set point range. The set point (expressed as % w/v) may be between about 0.1% and about 4%, at least about 0.01%, at least about 0.02%, at least about 0.04%, at least about 0.06%, at least about 0.08%, at least about 0.1%, at least about 0.15%, at least about 0.2%, at least about 0.25%, at least about 0.3%, at least about 0.35%, at least about 0.4%, at least about 0.45%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 1.2%, at least about 1.4%, at least about 1.6%, at least about 1.8%, at least about 2%, up to about 4%, up to about 3.75%, up to about 3.5%, up to about 3.25%, up to about 3%, up to about 2.75%, up to about 2.5%, up to about 2.25%, up to about 2%, up to about 1.75%, up to about 1.5%, up to about 1.25%, up to about 1%, between about 0.01% and about 4%, between about 0.02% and about 3.75%, between about 0.04% and about 3.5%, between about 0.08% and about 3.25%, between about 0.1% and about 3%, between about 0.2% and about 2.75%, between about 0.3% and about 2.5%, between about 0.4% and about 2.25%, between about 0.5% and about 2%, between about 0.6% and about 1.75%, between about 0.7% and about 1.5%, or between about 0.8% and about 1.25%. For example, the set point may be the same as the bolus concentration or within plus or minus 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%, 70%, 80%, 90% or 100% of the bolus concentration. 15 2016273912 14 Dec 2016 [00118] The ethanol concentration set point may be maintained by controlling ethanol production by yeast cells during fermentation. For example, the ethanol concentration may be increased by increasing the concentration of glucose (e.g., increasing the rate of glucose feed), decreasing the availability of oxygen, by decreasing the intensity of agitation (e.g., lowering the fermenter input power), decreasing the gas pressure in the fermenter, decreasing the flow rate of supplied air or other gas mixture, increasing the viscosity of the culture, or decreasing the concentration of oxygen in the supplied air or other gas mixture (e.g., if oxygen supplementation is being used). Ethanol production may also be increased by increasing the fermentation temperature. Likewise, the ethanol concentration may be decreased by decreasing the glucose concentration (e.g., decreasing the rate of glucose feed), decreased by increasing the availability of oxygen, by increasing the intensity of agitation (e.g., increasing the fermenter input power), increasing the gas pressure in the fermenter, increasing the flow rate of supplied air or other gas mixture, decreasing the viscosity of the culture, or increasing the concentration of oxygen in the supplied air or other gas mixture (e.g., if oxygen supplementation is being used). Ethanol production may also be decreased by decreasing the fermentation temperature.

[00119] Using the methods of the present disclosure, the relative abundance of undesired side-product(s) may be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% , at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or down to undetectable levels compared to initial abundance levels, relative to conventional methods. Exemplary undesired side-products whose relative abundance may be so decreased may include one or more species having a different apparent molecular weight than the desired multi-subunit complex. For example, apparent molecular weight may be affected by differences in stoichiometry, folding, complex assembly, and/or glycosylation. For example, such undesired side products may be detected using size exclusion chromatography and/or gel electrophoresis, and may have a higher or lower apparent molecular weight than the desired multi-subunit complex. In exemplary embodiments, the undesired side-products may be detected under reducing conditions. In other exemplary embodiments, the undesired side-products may be detected under non-reducing conditions.

[00120] In exemplary embodiments, the present disclosure also provides improved methods and compositions of matter that provide for the recombinant production of antibodies 16 2016273912 14 Dec 2016 and other multi-subunit complexes, with a higher yield. In exemplary embodiments, the yield may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or more (relative to conventional methods) using the methods disclosed herein.

[00121] In exemplary embodiments, the host cell in which the multi-subunit proteins may be produced may be a yeast, for example in a Pichia species such as P. pastoris or another methylotrophic yeast, or in a Saccharomyces species such as S. cerevisiae, or another yeast such as a Schizosaccharomyces (e.g., S. pombe). Other examples of methylotrophic yeast which may be utilized in the present invention include Pichia angusta (also known in the art as Hansenula polymorpha), Pichia guillermordii, Pichia methanolica, Pichia inositovera, Ogataea nitratoaversa, and Candida boidnii.

[00122] The host cell may be a eukaryotic cell, such as a yeast cell, such as a methylotrophic yeast, such as a yeast of the genus Pichia. Exemplary methylotrophic yeasts of the genus Pichia include Pichia pastoris, Pichia angusta, Pichia guillermordii, Pichia methanolica, and Pichia inositovera. The host cell may be produced by mating, e.g., by mating two haploid yeast cells that each contain one or more copies of at least one gene encoding a subunit of the multi-subunit complex.

[00123] In a preferred embodiment, the methylotrophic yeasts of the genus Pichia is Pichia pastoris. The host cell may be a diploid or tetraploid cell.

[00124] At least one of said genes encoding said subunits of the desired multi-subunit complex, such as said desired antibody light chain and / or heavy chain, may be expressed under control of an inducible or constitutive promoter, such as CUP1 (induced by the level of copper in the medium; see Koller et al., Yeast 2000; 16: 651-656.), tetracycline inducible promoters (see, e.g., Staib et al., Antimicrobial Agents And Chemotherapy, Jan. 2008, p. 146-156), thiamine inducible promoters, AOX1, ICL1, glyceraldehyde-3-phosphate dehydrogenase (GAP), FLD1, ADH1, alcohol dehydrogenase II, GAL4, PH03, PH05, and Pyk promoters, chimeric promoters derived therefrom, yeast promoters, mammalian promoters, insect promoters, plant promoters, reptile promoters, amphibian promoters, viral promoters, and avian promoters. 17 2016273912 14 Dec 2016 [00125] The host cell may secrete said desired multi-subunit complex into the culture medium. Alternatively or in addition, said desired multi-subunit complex may be retained in said host cell and may be isolated therefrom.

[00126] The desired multi-subunit complex may comprise an antibody, such as a monospecific or bispecific antibody. The antibody may be an antibody that specifically binds any antigen.

[00127] The desired multi-subunit complex may be an antibody other than any of the antibodies (e.g., an antibody other than any of the anti-NGF antibodies) disclosed in U.S. Provisional Application No. 61/418,832, filed December 1, 2010, PCT/US11/62963, filed December 1, 2011, U.S. Ser. No. 13/309,295, filed December 1, 2011, U.S. Ser. No. 13/309,153, filed December 1, 2011, U.S. Ser. No. 13/308,665 filed on December 1, 2011, and U.S. Ser. No. 13/308,831, filed December 1, 2011. In an exemplary embodiment, the desired multi-subunit complex may not be any of the following antibodies: Abl-NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, AblO-NGF, Abll-NGF, Abl2-NGF, AM3-NGF, AM4-NGF, AM5-NGF, AM6-NGF, AM7-NGF, AM8-NGF, AM9-NGF, Ab20-NGF, and Ab21-NGF. In a further exemplary embodiment, the desired multi-subunit complex may not be an Fab2 fragment of any of the following antibodies: Abl-NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, AblO-NGF, Abll-NGF, Abl2-NGF, AM3-NGF, Abl4-NGF, Abl5-NGF, Abl6-NGF, Abl7-NGF, Abl8-NGF, Abl9-NGF, Ab20-NGF, and Ab21-NGF. In a further exemplary embodiment, the desired multi-subunit complex may not be an Fabl fragment of any of the following antibodies: Abl-NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, AblO-NGF, Abll-NGF, Abl2-NGF, Abl3-NGF, Abl4-NGF, Abl5-NGF, Abl6-NGF, Abl7-NGF, Abl8-NGF, Abl9-NGF, Ab20-NGF, and Ab21-NGF. In a further exemplary embodiment, the desired multi-subunit complex may not comprise an antibody containing at least one, at least two, at least three, at least four, at least five, or at least all six of the complementarity determining regions (CDRs) contained in any of the following antibodies: Abl-NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, AblO-NGF, Abll-NGF, Abl2-NGF, Abl3-NGF, Abl4-NGF, Abl5-NGF, Abl6-NGF, Abl7-NGF, Abl8-NGF, Abl9-NGF, Ab20-NGF, or Ab21-NGF and optionally having binding 18 2016273912 14 Dec 2016 specificity for NGF. For example, the desired multi-subunit complex may not comprise or may not consist of the light and heavy chain polypeptide sequences SEQ ID NOs: 51 and 401, respectively, and/or SEQ ID NOs: 53 and 402, respectively, and/or SEQ ID NOs: 405 and 406, respectively, and/or SEQ ID NOs: 407 and 408, respectively. As a further example, the desired multi-subunit complex may not comprise an antibody containing at least one, at least two, at least three, at least four, at least five, or at least all six of the CDRs of SEQ ID NOs: 55, 56, 57, 58, 59, and 60, and optionally having binding specificity for NGF.

[00128] The desired multi-subunit complex may comprise an antibody of any type. Exemplary antibody types include antibodies of any mammalian species, e.g., human, mouse, rat, rabbit, goat, sheep, cow, etc. Preferably, the antibody is a human antibody or a humanized antibody that may be of rabbit origin. The desired antibody may be a monovalent, bivalent, or multivalent antibody.

[00129] At least one of said genes that provide for expression of a subunit of the desired multi-subunit complex, such as the light chain and/or heavy chain of a desired antibody, in at least one of said host cells in said panel may be optimized for expression in said host cell (e.g., by selecting preferred codons and/or altering the percentage AT through codon selection).

[00130] The purity of said desired multi-subunit complex, such as a desired antibody, may be assessed by measuring the fraction of the desired multi-subunit complex produced by said host cell that is non-glycosylated, is contained in complexes having the expected apparent hydrodynamic radius and/or apparent molecular weight (e.g., measured by size exclusion chromatography), has the expected electrophoretic mobility (e.g., detected by gel electrophoresis, such as SDS-PAGE, and optionally Western blotting), and / or by measuring the specific activity of the multi-subunit complex (e.g., specific binding a target of a desired antibody).

[00131] The desired multi-subunit complex may be an antibody, and yield of said antibody may be assessed by determining the amount of desired antibody produced by said host cell discounting any product-associated variants that are glycosylated, contained in antibody complexes other than complexes having the expected apparent molecular weight or hydrodynamic radius, and / or that fail to specifically bind to the target of said desired antibody. 19 2016273912 14 Dec 2016 [00132] The subject methods may produce a supernatant antibody titer of at least 100 mg / L, at least 150 mg / L, at least 200 mg / L, at least 250 mg / L, at least 300 mg / L, between 100 and 300 mg / L, between 100 and 500 mg / L, between 100 and 1000 mg / L or in excess of 1000 mg/L e.g., as high as 1200 mg/L, as high as 10,000 mg / L, or higher.

[00133] In another aspect, the host cell that produces a desired multi-subunit complex may be a diploid or tetraploid cell of the genus Pichia, such as a Pichia pastoris cell. The genes that provide for expression of the subunits of said desired multi-subunit complex, such as the light chain and heavy chain of a desired antibody, may be integrated into genome of said host cell, and/or may be contained on an extrachromosomal element, plasmid, or artificial chromosome.

[00134] In another aspect, the host cell that produces a desired multi-subunit complex may be engineered to increase yield and / or purity for example as further described in U.S. Provisional Application Ser. No. 61/525,307 (Atty. Docket No. 67858.730200), filed August 31, 2011, which is incorporated by reference herein in its entirety. As described therein, yield and purity of an antibody or other multi-subunit complex can be greatly improved by altering the number of copies per cell of the genes encoding each subunit. For example, where the desired multi-subunit complex is an antibody, the host cell may comprise more copies of the gene that provide for the expression of the light chain than copies of the gene that provide for expression of the heavy chain. In exemplary embodiments, the host cell may comprise from 1-10 copies of a gene encoding the light chain and from 1-10 copies of a gene encoding the heavy chain. The respective number of copies of the gene encoding the heavy chain and the number of copies of the gene encoding the light chain in said host cell may be: 2 and 2, 2 and 3, 3 and 3, 3 and 4, 3 and 5, 4 and 3, 4 and 4, 4 and 5, 4 and 6, 5 and 4, 5 and 5, 5 and 6, or 5 and 7, respectively. Additional exemplary combinations of heavy and light chain gene copy numbers include any combination of up to ten copies of the heavy and/or light chain gene, such as H2xLl, H3xLl, H4xLl, H5xLl, H6xLl, H7xLl, H8xLl, H9xLl, HlOxLl, HlxL2, H2xL2, H3xL2, H4xL2, H5xL2, H6xL2, H7xL2, H8xL2, H9xL2, H10xL2, HlxL3, H2xL3, H3xL3, H4xL3, H5xL3, H6xL3, H7xL3, H8xL3, H9xL3, H10xL3, HlxL4, H2xL4, H3xL4, H4xL4, H5xL4, H6xL4, H7xL4, H8xL4, H9xL4, H10xL4, HlxL5, H2xL5, H3xL5, H4xL5, H5xL5, H6xL5, H7xL5, H8xL5, H9xL5, H10xL5, HlxL6, H2xL6, H3xL6, H4xL6, H5xL6, H6xL6, H7xL6, H8xL6, H9xL6, H10xL6, HlxL7, H2xL7, H3xL7, H4xL7, H5xL7, H6xL7, H7xL7, H8xL7, H9xL7, 20 2016273912 14 Dec 2016 H10xL7, HlxL8, H2xL8, H3xL8, H4xL8, H5xL8, H6xL8, H7xL8, H8xL8, H9xL8, H10xL8, HlxL9, H2xL9, H3xL9, H4xL9, H5xL9, H6xL9, H7xL9, H8xL9, H9xL9, H10xL9, HlxLlO, H2xL10, H3xL10, H4xL10, H5xL10, H6xL10, H7xL10, H8xL10, H9xL10, HlOxLlO, where the number following the Ή” identifies the number of copies of the heavy chain gene, and the number following the “L” identifies the number of copies of the light chain gene. For example, the specified number of heavy and light chain gene copies may be tandemly integrated into a single locus, or into multiple loci (any or all of which may contain more than one copy). Optionally, each genomic locus may contain no more than three or four tandemly integrated gene copies, thereby promoting copy number stability during propagation and/or antibody production.

[00135] Culturing most typically involves proving cells with an energy source, oxygen, and nutrients. Methods are also known in the literature for design and optimization of P. pastoris fermentations for expression of recombinant proteins, including optimization of the cell density, broth volume, substrate feed rate, and the length of each phase of the reaction. See Zhang et al., “Rational Design and Optimization of Fed-Batch and Continuous Fermentations” in Cregg, J. M., Ed., 2007, Pichia Protocols (2nd edition), Methods in Molecular Biology, vol. 389, Humana Press, Totowa, N.J., pgs. 43-63. The culture may be provided with a gas mixture comprising oxygen, such as air with or without oxygen supplementation. The yeast culture may be cultured in a culture medium which may be a minimal medium, may lack selective agents, and / or may lack pre-formed amino acids or other complex biomolecules. The culture medium may also be a complex medium (e.g., containing yeast extract and/or plant peptone(s)). The medium may include a nitrogen source (e.g., methylamine chloride, NH4S04, yeast extract, soy peptone, other plant peptones, etc.). Exemplary minimal media include minimal dextrose medium (MD) (1.34% yeast nitrogen base (YNB) (w/o amino acids), 4 x 10_5% biotin, and 2% glucose.), buffered minimal glycerol complex medium (BMGY) (1% yeast extract, 2% peptone, 1% glycerol, 1.34% YNB (w/o amino acids), 4 x 10-5% biotin and 100 mM potassium phosphate (pH 6.0)). Media may include one or more salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as potassium phosphate, Tris, or HEPES), nucleosides (such as adenosine and thymidine), antibiotics (e.g., added to inhibit growth of contaminants and/or for maintenance of a selectable marker), trace elements, and glucose or another energy source. Any supplements and substitutions may also be included at appropriate concentrations that would be known to those skilled in the art. 21 2016273912 14 Dec 2016 [00136] The culture may be grown to a high cell density, such as at least 50 g/L, at least 100 g/L, at least 300 g/L, at least 400 g/L, at least 500 g/L, or at least 700 g/L. These culture densities are illustrative rather than limiting, and suitable culture densities may be readily determined by those of ordinary skill in the art.

[00137] The yeast cells may be cultured for at least 20 doublings and maintain high levels of expression of said antibody after said at least 20 doublings.

[00138] The yeast cells may be cultured for at least 50 doublings and maintain high levels of expression of said antibody after said at least 50 doublings.

[00139] The yeast cells may be cultured for at least 100 doublings and maintain high levels of expression of said antibody after said at least 100 doublings.

[00140] In another aspect, the present disclosure provides a culture medium containing a stable diploid Pichia yeast culture produced according to any of the foregoing methods, wherein the culture medium may comprise expression levels of said desired antibody which may be at least about 50 mg/liter, 100 mg/liter, 500 mg/liter, 750 mg/liter, 1000 mg/liter, 1250 mg/liter, 1500 mg/liter, 1750 mg/liter, 2000 mg/liter, or more. These yield values are illustrative rather than limiting. Optionally, yield may be optimized, for example using the methods and general approach described in Zhang et al. (2007), supra. For example, yield may be optimized by varying temperature, pH, media composition (e.g., carbon source, carbon source concentration, mixture of two or more carbon sources, nitrogen source and concentration, concentration of salts and nutrients including KH2PO4, K2HPO4, MgSCL, potassium sulfate, sodium citrate, potassium sulfate, sodium citrate, trace metals such as cobalt chloride, cupric sulfate, sodium iodide, manganese sulfate, sodium molybdate, boric acid, zinc chloride, ferrous sulfate, vitamins such as biotin, inositol, thiamine, peptone, yeast extract, casamino acids, urea, ammonium phosphate or other ammonium ions, L-arginine-hydrochloride), time, culture density, oxygenation, and other factors that influence yield. For example, yield, expression, and/or purity of the desired multisubunit complex may in some instances be improved by maintaining the temperature at a desired set point, e.g., a set point between about 15°C and about 30 °C, such as between about 17 °C and about 25 °C). Without intent to be limited by theory, it is hypothesized that controlling the temperature may assist intracellular trafficking through the folding and post-translational 22 2016273912 14 Dec 2016 processing pathways, and/or may decrease the activity of cellular proteases. Likewise, yield, expression, and/or purity of the desired multi-subunit complex may in some instances be improved by maintaining the pH of the culture medium at a desired set point, e.g., a set point between pH 3 to pH 8, such as between pH 4 and pH 7.

[00141] In another aspect, the present disclosure provides a culture medium containing a stable diploid Pichia pastoris yeast culture produced according to any of the foregoing methods that expresses said desired antibody into a culture medium wherein the cell density of said diploid cells in said culture may be at least about 50 g/L, 100 g/L, 300 g/L, 400 g/L, 500 g/L, 700 g/L or more. These culture densities are illustrative rather than limiting, and suitable culture densities may be readily determined by those of ordinary skill in the art.

[00142] At least one subunit of said antibody or other multi-subunit protein may comprise a secretion signal, such as the S. chicken lysozyme (CLY) signal peptide; CLY-L8; S. cerevisiae invertase (SUC2) signal peptide; MF-alpha (Prepro); MF-alpha (Pre)-apv; MF-alpha (Pre)-apv-SLEKR; MF-alpha (Prepro)-(EA)3; aF signal peptide; KILM1 signal peptide; repressible acid phosphatase (PHOl) signal peptide; A. niger GOX signal peptide; Schwanniomyces occidentalis glucoamylase gene (GAM1) signal peptide; human serum albumin (HSA) signal peptide without pro-sequence; human serum albumin (HSA) signal peptide with pro-sequence; ISN signal peptide; IFN signal peptide; HGH signal peptide; phytohaemagglutinin (PHA); Silkworm lysozyme; Human lysozyme (LYZ1); activin receptor type-1; activin type II receptor; P. pastoris immunoglobulin binding protein (PpBiP); human antibody 3D6 light chain leader; and any combination thereof.

[00143] The host cell may be produced by mating two haploid yeast cells that each contain one or more copies of a gene encoding one or more subunits of said antibody or other multisubunit protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[00144] FIG. 1A-B. Purity of a recombinantly produced Ab-A was improved by a bolus addition of ethanol prior to the start of a glucose feed in yeast cultures from which the antibodies were produced. Antibodies were harvested after 97 hours of culture and purified by protein-A affinity, then purity was assessed by SDS-PAGE using a non-reduced gel (FIG. 1 A) to resolve 23 2016273912 14 Dec 2016 the desired full antibody (arrow, “Full Ab (H2L2)”) from undesired product-associated variants. Complexes having aberrant stoichiometry were identified based on their molecular weight, affinity for protein A, and additional studies further described below, as a “half antibody” species containing one heavy and one light chain (arrow, “HILI”) and a complex containing two heavy chains and one light chain (“H2L1”). The relative abundance of the H2L1 and H1L1 complexes was greatly decreased by the bolus addition of ethanol during antibody production. Compare FIG. 1A, lanes 2-3 (no bolus) to lane 5 (with bolus). FIG. IB shows the same samples processed under reducing conditions, which separated each of the full antibody, H1L1, and H2L1 complexes into individual heavy and light chains, confirming that the H1L1 and H2L1 complexes are composed of full-length heavy and light chains. Lane order in FIGS. 1A-B: Lane 1: molecular weight marker; lanes 2 and 3: control samples prepared from fermentation cultures without a bolus addition of ethanol; lane 4: no sample; lane 5: sample prepared from fermentation cultures with a bolus addition of ethanol.

[00145] FIGS. 1C-E show the gel band density plotted along the length of the non-reduced gel (FIG. 1 A, lanes 2, 3, and 5, respectively); arrows identify the peaks corresponding to the H1L1 species. FIG. IF tabulates the area contained in the H1L1 peaks shown in FIGS. 1C-E, demonstrating approximately 90% reduction in the relative abundance of the H1L1 complexes. H2L1 complex abundance was not quantified due to the incomplete resolution from the full antibody peaks.

[00146] FIGS. 2A-B and 3A-B demonstrate reproducibility of the improvement in purity of Ab-A by a bolus addition of ethanol to the yeast cultures. Antibodies were harvested after 87 or 86 hours of culture (FIGs. 2 and 3, respectively) and purified by protein-A affinity, then purity was assessed by SDS-PAGE using a non-reduced gel. The abundance of the H1L1 and H2L1 complexes (arrows) were again decreased by the bolus addition of ethanol. Compare FIG. 2A, lane 3 (no bolus) to lane 2 (with bolus), and FIG. 3 A, lanes 4-6 (no bolus) with lanes 2 and 4 (with bolus). FIG. 2B shows the same samples as in FIG. 2A processed under reducing conditions, again confirming that the observed product-associated variants are composed of full-length heavy and light chains. Lane order in FIGS. 2A-B: Lane 1: molecular weight marker; lane 2: sample prepared from a fermentation culture with a bolus addition of ethanol; lane 3: control sample prepared from a fermentation culture with no bolus addition of ethanol. Lane 24 2016273912 14 Dec 2016 order in FIG. 3 A: Lane 1: molecular weight marker; lanes 2 and 4: samples prepared from fermentation cultures with a bolus addition of ethanol; lane 3: no sample; lanes 5-7: control samples prepared from fermentation cultures with no bolus addition of ethanol.

[00147] FIGS. 2C and 2D show the gel band density plotted along the length of the non-reduced gel (FIG. 2A, lanes 2, and 3, respectively); arrows identify the peaks corresponding to the H1L1 species. FIGS. 2E and 3B tabulate the area contained in the H1L1 peaks shown in FIGS. 2C and FIG. 3A, demonstrating approximately 85% reduction in the relative abundance of H1L1 complexes in FIG. 2A and approximately 87% average reduction in the relative abundance of H1L1 complexes in FIG. 3A.

[00148] FIG. 4A-D. Purity of a second recombinant antibody (”Ab-B”) was also improved by a bolus addition of ethanol prior to the production phase of a fermentation process. Samples of fermentation culture broth were harvested after 67 hours (“T67) or 87 hours (“T87”) of culture (FIGs. 4A-B and 4C-D, respectively) and antibodies were purified by protein-A affinity. Purity was then assessed by SDS-PAGE using non-reduced gels (FIGS. 4A and 4C).

At both assessed time points, the abundance of the half-antibody species (H1L1) and the H2L1 complex was greatly decreased in fermentation cultures prepared that received a bolus addition of ethanol, relative to control cultures that did not receive a bolus addition of ethanol. Compare FIG. 4A, lanes 2-3 (no bolus) to lanes 6-7 (with bolus), and FIG. 4C, lanes 2-3 (no bolus) to lanes 6-7 (with bolus). FIGs. 4B and 4D shows the same samples processed under reducing conditions. Lane order in FIGS. 4A-D: Lane 1: molecular weight marker; lanes 2-3: control sample prepared from fermentation cultures with no bolus addition of ethanol; lanes 4-5: no sample; lanes 6-7: samples prepared from fermentation cultures with a bolus addition of ethanol.

[00149] FIGS. 4E and 4F tabulate the area contained in the H1L1 peaks shown in FIGS. 4A (T67) and 4C (T87), respectively, demonstrating that the bolus addition of ethanol produced about a 73% reduction in the relative abundance of H1L1 complexes at the earlier time point shown FIG. 4A and about a 34% average reduction in the relative abundance of H1L1 complexes at the later time point shown in FIG. 4C.

[00150] FIG. 5A-B. Purity of a third recombinant antibody (Ab-C) was also improved by a bolus addition of ethanol prior to the production phase of fermentation. Antibodies were 25 2016273912 14 Dec 2016 harvested after 86 hours of culture and purified by protein-A affinity, then purity was assessed by SDS-PAGE using a non-reduced gel (FIGS. 5A). The H1L1 and H2L1 complexes were less abundant in the Ab-C product even without the addition of a bolus of ethanol, leaving less room for improvement. Nonetheless, the abundance of the half-antibody species (H1L1) and the H2L1 complex was noticeably decreased in fermentation cultures that received a bolus addition of ethanol, relative to control cultures that did not receive a bolus addition of ethanol. Compare FIG. 5A, lanes 5-6 (no bolus) to lane 3 (with bolus). FIG. 5B shows the same samples processed under reducing conditions. Lane order in FIGS. 5A-B: Lane 1: molecular weight marker; lane 2: no sample; lane 3: sample prepared from fermentation cultures that received a bolus addition of ethanol; lane 4: no sample; lanes 5-6: control sample prepared from fermentation cultures that did not receive a bolus addition of ethanol.

[00151] FIG. 5C tabulates the area contained in the HI LI peaks shown in FIGS. 5 A, demonstrating about a 61% average reduction in the relative abundance of H1L1 complexes by the bolus addition of ethanol.

[00152] FIG. 6A-F shows assessment of the relative purity of the Ab-A preparations shown in FIGS. 1-3 by size exclusion chromatography. In each panel, the main peak contains the full antibody containing two heavy and two light chains (H2L2). The H1L1 species was not resolved from the main peak by this method (thought to be due H1L1 dimers forming by non-covalent association which is retained under the conditions used). However, other undesired product-associated variants were detected, including higher molecular weight species (left of main peak) and lower molecular weight species (right of main peak). A prominent peak though to correspond to antibody dimers containing two full antibodies (H4L4) was detected (arrow) and the relative abundance of these was decreased in samples prepared from fermentation cultures that received a bolus addition of ethanol. Compare FIGS. 6A, 6C, and 6E (no bolus) to FIGS. 6B, 6D, and 6F (with bolus).

[00153] FIG. 7 summarizes quantitation of the amount of product-associated variants detected by SEC for the six Ab-A samples shown in FIG. 6 and five additional samples. For each identified sample (col. 1), the run set number (col. 2, identifying fermentation runs that were conducted in parallel), bolus added (either lOg/L or none, col. 3), and elapsed culture time 26 2016273912 14 Dec 2016 before culture samples were taken and processed (col. 4) are shown, together with the fraction of protein detected in the main peak (“SEC Main Peak %,” col. 5). The bolus addition of ethanol at the end of the growth phase increased the average percentage contained in the main peak, from 80.3% up to 90.6%.

[00154] FIG. 8 summarizes quantitation of the amount of product-associated variants detected by SEC for Ab-B antibody samples shown in FIG. 4. For each fermentation run (col. 1), the bolus added at the end of the growth phase (either lOg/F or none, col. 2), and elapsed culture time before culture samples were taken and processed (col. 3) are shown, together with the fraction of protein detected in the main peak (“SEC Main Peak %,” col. 4). Overall purity was increased, with the main peak increasing from 76% to 79% at T67 and from 60% to 73% at T87.

[00155] FIG. 9 summarizes quantitation of the amount of product-associated variants detected by SEC for the Ab-C antibody samples shown in FIG. 5. For each identified fermentation run (col. 1), the bolus added (either lOg/F or none, col. 2), and elapsed culture time before the sample was taken and processed (col. 3) are shown, together with the fraction of protein detected in the main peak (“SEC Main Peak %,” col. 4). There was little difference in overall purity as detected by this method, with about 89% of product contained in main peak with or without the ethanol bolus. This was apparently due to the high initial purity of the Ab-C antibody even without the bolus addition. Additionally, SEC did not resolve the HI El species from the full antibody and accordingly the decreased production of this species due to the ethanol bolus was not reflected in SEC results.

[00156] FIG. 10 summarizes the results of mass spectrometry measurement of the quantity of a free heavy chain (lacking a disulfide bond to a second heavy chain) in Ab-A antibody samples containing high or low amounts of the HIE 1 band. As expected, the amount of free heavy chain correlated with the amount of the HIE 1 band, confirming the identity thereof as containing one heavy and one light chain and lacking a disulfide bond to a second heavy chain.

[00157] FIGS. 11-13 show the correlation between addition of an ethanol bolus and cell viability. The addition of an ethanol bolus generally improved cell viability and antibody purity for the Ab-A antibody (FIG. 11) and the Ab-B antibody (FIG. 12). These results suggest that the 27 2016273912 14 Dec 2016 improvement in cell viability may account for at least part of the improvement in antibody purity from the ethanol bolus addition. Consistent with these results, the Ab-C antibody culture exhibited greater antibody purity and cell viability than the Ab-A and Ab-B cultures (FIG. 13). Apparently because the viability of the Ab-C antibody-producing cultures was already high in these experiments, there was little room for improvement and the cultures exhibited little improvement in viability from the bolus addition of ethanol. In FIGS. 11-13, filled bars indicate no bolus, while open bars indicate a bolus addition of ethanol. Viability was determined from fermentation cultures sampled with 1.5 hours of the time at which samples were collected for purity analyses (as identified in the preceding slides).

[00158] FIG. 14 shows that a wide range of ethanol bolus concentrations can produce the same improvement in antibody purity. Ab-A was produced with a bolus addition of ethanol between 5 g/L (0.5% w/v) and 15 g/L (1.5% w/v) and purified by protein A affinity, then the purity was analyzed by non-reduced SDS-PAGE. Each culture exhibited similarly low levels of the H2L1 and H1L1 complexes at 63 hours (FIG. 14A) and 86 hours (FIG. 14B). Lane order in FIGS. 14A-B: lane 1: molecular weight markers; lanes 2 and 7: 5 g/L bolus; lanes 3 and 5:10 g/L bolus; lanes 4 and 6: 15 g/L bolus.

[00159] FIG. 15 shows that the time elapsed between the dissolved oxygen spike and the ethanol bolus addition can vary considerably while giving similar improvement in antibody purity. Ab-A was produced with a bolus addition of ethanol of 10 g/L (1% w/v) and purified by protein A affinity, then the purity was analyzed by non-reduced SDS-PAGE (FIG. 15A). The “starvation period,” the time between the dissolved oxygen spike (indicating exhaustion of the carbon source in the culture) and the bolus addition of ethanol, was varied between 0 and 3 hours. Each culture exhibited similarly low levels of the H2L1 and H1L1 complexes irrespective of the duration of the starvation period, indicating that antibody purity is relatively insensitive to absence of a starvation period or a starvation period of at least up to three hours. The same samples were analyzed on a reduced gel (FIG. 15B). Lane order in FIGS. 15A-B: lane 1: molecular weight markers; lanes 2-4: no sample; lane 5: 0 hours starvation period; lane 6: 3 hours starvation period. 28 2016273912 14 Dec 2016 [00160] FIG. 16 shows the effect of the equilibration period (the time between ethanol bolus addition and feed start) on antibody purity. The Ab-B antibody was produced with a bolus addition of ethanol of 10 g/L (1% w/v) and purified by protein A affinity, then the purity was analyzed by non-reduced SDS-PAGE (FIG. 16A). The duration of the equilibration period was either 0, 30, or 60 minutes. The 60 minute equilibration period resulted in a lower antibody purity (higher abundance of the H2L1 and H1L1 complexes). Culture viability was also markedly lower with a 60 minute equilibration period, particularly earlier in the culture (at 23 hours, FIG. 16B); viability had improved somewhat by the end of the culture (at 85 hours, FIG. 16C). Lane order in FIG. 16A: lane 1: molecular weight markers; lanes 2 and 4: no sample; lane 3: 30 minutes equilibration; lanes 5 and 6: 60 minutes equilibration time; lanes 7 and 8: 0 minutes equilibration time. In FIGS. 16B-C, filled bars indicate an equilibration period of zero minutes, hatched bars indicate an equilibration period of 30 minutes and open bars indicate an equilibration period of 60 minutes.

DETAILED DESCRIPTION

[00161] Applicants have unexpectedly discovered that purity of multi-subunit complexes expressed from yeast can be greatly improved by addition of a bolus of ethanol to the culture media. A single bolus addition of ethanol was demonstrated to improve purity over a sustained period of production, for up to at least 97 hours.

[00162] The present disclosure provides improved methods and compositions of matter that provide for the recombinant production of antibodies and other multi-subunit complexes with increased purity and decreased production of one or more undesired side-products. In exemplary embodiments, relative to the desired multi-subunit complex the undesired side product(s) may exhibit one or more of: altered stoichiometry, aberrant glycosylation, differences in apparent molecular weight, differences in disulfide bonds, differences in hydrodynamic radius, fragments and/or truncated forms of one or more subunits. Undesired side-products may exhibit one or more additional differences as well. Undesired side-products may also be detected by their effects on a preparation, e.g., alteration in the level of specific activity, immunogenicity, or other effects on physical constitution and/or function of the desired multi-subunit complex. 29 2016273912 14 Dec 2016 [00163] For example, when the desired multi-subunit complex is an antibody, the undesired side products may include an H1L1 or “half antibody” species (i.e., containing a heavy chain and a light chain, wherein the heavy chain is not linked by a disulfide bond to another heavy chain), and/or a H2L1 species (i.e., containing two heavy chains and one light chain, but lacking a second light chain).

[00164] Though not intending to be limited by theory, it is hypothesized that a rapid increase in ethanol concentration (which can be brought about by a bolus addition of ethanol) can cause sustained changes in gene expression which confer a lasting improvement in the production of properly folded and assembled multi-subunit complexes and/or increases processing of improperly folded or misassembled multi-subunit complexes, leading to improved purity in the multi-subunit complex. Additionally, it was demonstrated that the improved antibody purity correlated with improved viability of the yeast in the culture, and based thereon Applicants hypothesize that the improved viability may account (at least in part) for the improved purity, though this theory is not intended to be limiting.

[00165] In a preferred embodiment, the heterologous multi-subunit complex is an antibody or antibody fragment, such as a humanized antibody, comprised of two heavy chain subunits and two light chain subunits. Preferred host cells include yeasts, and particularly preferred yeasts include methylotrophic yeast strains, e.g., Pichia pastoris, Hansenula polymorpha {Pichia angusta), Pichia guillermordii, Pichia methanolica, Pichia inositovera, and others {see, e.g., U.S. Patent 4,812,405, 4,818,700, 4,929,555, 5,736,383, 5,955,349, 5,888,768, and 6,258,559 each of which is incorporated by reference in its entirety). The host cell may be produced by methods known in the art, such as transformation, mating, sporulation, etc.

[00166] In a preferred embodiment, the host cell may comprise more than one copy of one or more of the genes encoding the heterologous protein subunits. For example, multiple copies of a subunit gene may be integrated in tandem into one or more chromosomal loci. Tandemly integrated gene copies are preferably retained in a stable number of copies during culture for the production of the multi-subunit complex. For example, in the examples described below, gene copy numbers were generally stable for P. pastoris strains containing three to four tandemly integrated copies of light and heavy chain antibody genes. 30 2016273912 14 Dec 2016 [00167] One or more of the genes encoding the heterologous protein subunits are preferably integrated into one or more chromosomal loci of a host cell. Any suitable chromosomal locus may be utilized for integration, including intergenic sequences, promoters sequences, coding sequences, termination sequences, regulatory sequences, etc. Exemplary chromosomal loci that may be used in P. pastoris include PpURA5; OCH1; AOX1; HIS4; and GAP. The encoding genes may also be integrated into one or more random chromosomal loci rather than being targeted. In preferred embodiments, the chromosomal loci are selected from the group consisting of the pGAP locus, 3’ AOX TT, and the HIS4 TT locus. In additional exemplary embodiments, the genes encoding the heterologous protein subunits may be contained in one or more extrachromosomal elements, for example one or more plasmids or artificial chromosomes.

[00168] In exemplary embodiments, the multi-subunit protein may comprise two, three, four, five, six, or more non-identical subunits. Additionally, each subunit may be present one or more times in each multi-subunit protein. For example, the multi-subunit protein may be a multi-specific antibody such as a bi-specific antibody comprising two non-identical light chains and two non-identical heavy chains.

[00169] The subunits may be expressed from monocistronic genes, polycistronic genes, or any combination thereof. Each polycistronic gene may comprise multiple copies of the same subunit, or may comprise one or more copies of each different subunit.

[00170] Exemplary methods that may be used for manipulation of Pichia pastoris (including methods of culturing, transforming, and mating) are disclosed in Published Applications including U.S. 20080003643, U.S. 20070298500, and U.S. 20060270045, and in Higgins, D. R., and Cregg, J. M., Eds. 1998. Pichia Protocols. Methods in Molecular Biology. Humana Press, Totowa, N.J., and Cregg, J. M., Ed., 2007, Pichia Protocols (2nd edition), Methods in Molecular Biology. Humana Press, Totowa, N.J., each of which is incorporated by reference in its entirety.

[00171] An exemplary expression cassette that may be utilized is composed of the glyceraldehyde dehydrogenase gene (GAP gene) promoter, fused to sequences encoding a secretion signal, followed by the sequence of the gene to be expressed, followed by sequences 31 2016273912 14 Dec 2016 encoding a P. pastoris transcriptional termination signal from the P. pastoris alcohol oxidase I gene (AOX1). The Zeocin resistance marker gene may provide a means of enrichment for strains that contain multiple integrated copies of an expression vector in a strain by selecting for transformants that are resistant to higher levels of Zeocin. Similarly, G418 or Kanamycin resistance marker genes may be used to provide a means of enrichment for strains that contain multiple integrated copies of an expression vector in a strain by selecting for transformants that are resistant to higher levels of Geneticin or Kanamycin.

[00172] Host strains that may be utilized include auxotrophic P. pastoris or other Pichia strains, for example, strains having mutations in metl, lys3, ura3 and adel or other auxotrophy-associated genes. Preferred mutations are incapable of giving rise to revertants at any appreciable frequency and are preferably partial or even more preferably full deletion mutants. Preferably, prototrophic diploid or tetraploid strains are produced by mating a complementing sets of auxotrophic strains.

[00173] Transformation of haploid P. pastoris strains and genetic manipulation of the P. pastoris sexual cycle may be performed as described in Pichia Protocols (1998, 2007), supra.

[00174] Prior to transformation, each expression vector may be linearized by restriction enzyme cleavage within a region homologous to the target genomic locus (e.g., the GAP promoter sequence) to direct the integration of the vectors into the target locus in the host cell. Samples of each vector may then be individually transformed into cultures of the desired strains by electroporation or other methods, and successful transformants may be selected by means of a selectable marker, e.g., antibiotic resistance or complementation of an auxotrophy. Isolates may be picked, streaked for single colonies under selective conditions and then examined to confirm the number of copies of the gene encoding the subunit of the multi-subunit complex (e.g., a desired antibody) by Southern Blot or PCR assay on genomic DNA extracted from each strain. Optionally, expression of the expected subunit gene product may be confirmed, e.g., by FACS, Western Blot, colony lift and immunoblot, and other means known in the art. Optionally, haploid isolates are transformed additional times to introduce additional heterologous genes, e.g., additional copies of the same subunit integrated at a different locus, and / or copies of a different subunit. The haploid strains are then mated to generate diploid strains (or strains of higher 32 2016273912 14 Dec 2016 ploidy) able to synthesize the multi-protein complex. Presence of each expected subunit gene may be confirmed by Southern blotting, PCR, and other detection means known in the art.

Where the desired multi-protein complex is an antibody, its expression may also be confirmed by a colony lift/immunoblot method (Wung et al. Biotechniques 21 808-812 (1996) and / or by FACS.

[00175] This transformation protocol is optionally repeated to target a heterologous gene into a second locus, which may be the same gene or a different gene than was targeted into the first locus. When the construct to be integrated into the second locus encodes a protein that is the same as or highly similar to the sequence encoded by the first locus, its sequence may be varied to decrease the likelihood of undesired integration into the first locus. For example, the sequence to be integrated into the second locus may have differences in the promoter sequence, termination sequence, codon usage, and/or other tolerable sequence differences relative to the sequence integrated into the first locus.

[00176] To mate P. pastoris haploid strains, each strain to be crossed can be patched together onto mating plates. For example, multiple matings can be conveniently performed at the same time by streaking each strain to be mated across a plate suitable for its growth, and the mating partners may be streaked across a second plate (preferably the plates are rich media such as YPD). Typically, after one or two days incubation at 30° C., cells from the two plates can be replica plated in a crisscross fashion onto a mating plate, resulting in a cross-hatched pattern with each pair of strains being co-plated and having the opportunity to mate at the intersection of a pair of the original streak lines. The mating plate can then be incubated (e.g., at 30° C.) to stimulate the initiation of mating between strains. After about two days, the cells on the mating plates can be streaked, patched, or replica plated onto media selective for the desired diploid strains (e.g., where the mated strains have complementary autotrophies, drop-out or minimal medium plates may be used). These plates can be incubated (e.g., at 30° C.) for a suitable duration (e.g., about three days) to allow for the selective growth of the desired diploid strains. Colonies that arise can be picked and streaked for single colonies to isolate and purify each diploid strain. 33 2016273912 14 Dec 2016 [00177] Expression vectors for use in the methods of the invention may further include yeast specific sequences, including a selectable auxotrophic or drug marker for identifying transformed yeast strains. A drug marker may further be used to amplify copy number of the vector in a yeast host cell, e.g., by culturing a population of cells in an elevated concentration of the drug, thereby selecting transformants that express elevated levels of the resistance gene.

[00178] In an exemplary embodiment, one or more of the genes encoding the heterologous protein subunits are coupled to an inducible promoter. Suitable exemplary promoters include the alcohol oxidase 1 gene promoter, formaldehyde dehydrogenase genes (FLD; see U.S. Pub. No. 2007/0298500), and other inducible promoters known in the art. The alcohol oxidase 1 gene promoter, is tightly repressed during growth of the yeast on most common carbon sources, such as glucose, glycerol, or ethanol, but is highly induced during growth on methanol (Tschopp et al., 1987; U.S. Pat. No. 4,855,231 to Stroman, D. W., et al). For production of foreign proteins, strains may be initially grown on a repressing carbon source to generate biomass and then shifted to methanol as the sole (or main) carbon and energy source to induce expression of the foreign gene. One advantage of this regulatory system is that P. pastoris strains transformed with foreign genes whose expression products are toxic to the cells can be maintained by growing under repressing conditions.

[00179] In another exemplary embodiment, one or more of the heterologous genes may be coupled to a regulated promoter, whose expression level can be upregulated under appropriate conditions. Exemplary regulated promoters include the CUP1 promoter (induced by the level of copper in the medium), tetracycline inducible promoters, thiamine inducible promoters, the AOX1 promoter, and the FLD1 promoter.

[00180] Though much of the present disclosure describes production of antibodies, the methods described herein are readily adapted to other multi-subunit complexes as well. Without intent to be limited by theory, it is believed that the yield and purity of multi-subunit complexes can be greatly influenced by the concentration and stoichiometry of the subunits, which are in turn influenced by the level of expression of the genes responsible for production of each subunit. The methods disclosed herein may readily be utilized to improve the yield and / or purity of any recombinant multi-subunit complex comprising two or more different subunits. 34 2016273912 14 Dec 2016

Additionally, the present methods are not limited to production of multi-protein complexes but may also be readily adapted for use with ribonucleoprotein (RNP) complexes including telomerase, hnRNPs, Ribosomes, snRNPs, signal recognition particles, prokaryotic and eukaryotic RNase P complexes, and any other complexes that contain multiple distinct protein and / or RNA subunits. The host cell that expresses the multi-subunit complex may be produced by methods known in the art. For example, a panel of diploid or tetraploid yeast cells containing differing combinations of gene copy numbers may be generated by mating cells containing varying numbers of copies of the individual subunit genes (which numbers of copies preferably are known in advance of mating).

Definitions [00181] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[00182] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

[00183] Bolus addition: In the present disclosure, “bolus addition” generally refers to rapid change in concentration of a substance (such as ethanol) in contact with cultured cells (for example, in a culture medium). For example, the substance may be added to the cultured cells in a single addition, a succession of more than one addition, and/or infused over a period of time (e.g., over about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 90, or 120 minutes). The substance may also be added by replacing the culture medium in part or in full, for example by concentrating the cells (using centrifugation, filtration, settling, or other methods), removing part or all of the medium, and adding the substance, or by adding the cells to a medium containing the 35 2016273912 14 Dec 2016 substance. The substance may be admixed with a carrier (e.g., culture media, water, saline, etc.). For example, a bolus addition of ethanol may comprise the addition of pure or concentrated ethanol (e.g., 100%, 95%, 70%, 50%, 60%, 40%, 30%, 20%, etc.) to the culture medium in an amount sufficient to produce the desired concentration. As another example, the cells may be added to a medium containing ethanol, e.g., by adding an inoculum containing the cells to a medium containing ethanol.

[00184] Bolus concentration: In the present disclosure, “bolus concentration” generally refers to the concentration that results from a bolus addition of a substance (e.g., ethanol).

[00185] Mating competent yeast species: In the present invention this is intended to broadly encompass any diploid or tetraploid yeast which can be grown in culture. Such species of yeast may exist in a haploid, diploid, or other polyploid form. The cells of a given ploidy may, under appropriate conditions, proliferate for an indefinite number of generations in that form. Diploid cells can also sporulate to form haploid cells. Sequential mating can result in tetraploid strains through further mating or fusion of diploid strains. The present invention contemplates the use of haploid yeast, as well as diploid or other polyploid yeast cells produced, for example, by mating or fusion (e.g., spheroplast fusion).

[00186] In one embodiment of the invention, the mating competent yeast is a member of the Saccharomycetaceae family, which includes the genera Arxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera; Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora; Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces. Other types of yeast potentially useful in the invention include Yarrowia; Rhodosporidium; Candida', Hansenula; Filobasium; Sporidiobolus\ Bullera; Leucosporidium and Filobasidella.

[00187] In a preferred embodiment of the invention, the mating competent yeast is a member of the genus Pichia or is another methylotroph. In a further preferred embodiment of the invention, the mating competent yeast of the genus Pichia is one of the following species: Pichia pastoris, Pichia methanolica, and Hansenula polymorpha (Pichia angusta). In a particularly preferred embodiment of the invention, the mating competent yeast of the genus Pichia is the species Pichia pastoris. 36 2016273912 14 Dec 2016 [00188] Haploid Yeast Cell·. A cell having a single copy of each gene of its normal genomic (chromosomal) complement.

[00189] Polyploid Yeast Cell·. A cell having more than one copy of its normal genomic (chromosomal) complement.

[00190] Diploid Yeast Cell·. A cell having two copies (alleles) of essentially every gene of its normal genomic complement, typically formed by the process of fusion (mating) of two haploid cells.

[00191] Tetraploid Yeast Cell: A cell having four copies (alleles) of essentially every gene of its normal genomic complement, typically formed by the process of fusion (mating) of two diploid cells. Tetraploids may carry two, three, four, or more different expression cassettes. Such tetraploids might be obtained in S. cerevisiae by selective mating homozygotic heterothallic a/a and alpha/alpha diploids and in Pichia by sequential mating of haploids to obtain auxotrophic diploids. For example, a [met his] haploid can be mated with [ade his] haploid to obtain diploid [his]; and a [met arg] haploid can be mated with [ade arg] haploid to obtain diploid [arg]; then the diploid [his] can be mated with the diploid [arg] to obtain a tetraploid prototroph. It will be understood by those of skill in the art that reference to the benefits and uses of diploid cells may also apply to tetraploid cells.

[00192] Yeast Mating: The process by which two yeast cells fuse to form a single yeast cell. The fused cells may be haploid cells or cells of higher ploidy (e.g., mating two diploid cells to produce a tetraploid cell).

[00193] Meiosis: The process by which a diploid yeast cell undergoes reductive division to form four haploid spore products. Each spore may then germinate and form a haploid vegetatively growing cell line.

[00194] Selectable Marker: A selectable marker is a gene or gene fragment that confers a growth phenotype (physical growth characteristic) on a cell receiving that gene as, for example through a transformation event. The selectable marker allows that cell to survive and grow in a selective growth medium under conditions in which cells that do not receive that selectable marker gene cannot grow. Selectable marker genes generally fall into several types, including 37 2016273912 14 Dec 2016 positive selectable marker genes such as a gene that confers on a cell resistance to an antibiotic or other drug, temperature when two temperature sensitive (“ts”) mutants are crossed or a ts mutant is transformed; negative selectable marker genes such as a biosynthetic gene that confers on a cell the ability to grow in a medium without a specific nutrient needed by all cells that do not have that biosynthetic gene, or a mutagenized biosynthetic gene that confers on a cell inability to grow by cells that do not have the wild type gene; and the like. Suitable markers include but are not limited to: ZEO; NEO (G418); LYS3; MET1; MET3a; ADE1; ADE3; URA3; and the like.

[00195] Integrated: A genetic element (typically a heterologous genetic element) that are covalently joined into a chromosome of an organism.

[00196] Tandemly integrated: Two or more copies of a genetic element that are integrated in adjacent locations in a chromosome. The two or more copies do not necessarily have the orientation; e.g., for transcribed genes, some copies may be transcribed from the Watson strand and others from the Crick strand.

[00197] Host cell: In the context of the present disclosure, the term host cell refers to a cell (e.g., a eukaryotic cell, such as a Pichia cell) which contains a heterologous gene. For example, the heterologous gene may provide for the expression of a subunit of a desired multisubunit complex, a gene involved in protein folding (e.g., a chaperone), expression, or secretion, and/or another desired gene. The heterologous gene may be integrated into the genome of the eukaryotic cell or contained in extrachromosomal element such as a plasmid or artificial chromosome.

[00198] Expression Vector. These DNA vectors contain elements that facilitate manipulation for the expression of a foreign protein within the target host cell. Conveniently, manipulation of sequences and production of DNA for transformation is first performed in a bacterial host, e.g. E. coli, and usually vectors will include sequences to facilitate such manipulations, including a bacterial origin of replication and appropriate bacterial selection marker. Selection markers encode proteins necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes 38 2016273912 14 Dec 2016 encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media. Exemplary vectors and methods for transformation of yeast are described, for example, in Burke, D., Dawson, D., & Steams, T. (2000). Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, which is incorporated by reference herein in its entirety.

[00199] Expression vectors for use in the methods of the invention may further include yeast specific sequences, including a selectable auxotrophic or drug marker for identifying transformed yeast strains. A drug marker may further be used to select for amplification of copy number of the vector in a yeast host cell.

[00200] The polypeptide coding sequence of interest is typically operably linked to transcriptional and translational regulatory sequences that provide for expression of the polypeptide in yeast cells. These vector components may include, but are not limited to, one or more of the following: an enhancer element, a promoter, and a transcription termination sequence. Sequences for the secretion of the polypeptide may also be included, e.g. a signal sequence, and the like. A yeast origin of replication is optional, as expression vectors are often integrated into the yeast genome.

[00201] Though optional, in one embodiment of the invention, one or more subunit of the multi-subunit complex is operably linked, or fused, to a secretion sequence that provides for secretion of the expressed polypeptide into the culture media, which can facilitate harvesting and purification of the heterologous multi-subunit complex. Even more preferably, the secretion sequences provide for optimized secretion of the polypeptide from the host cells (e.g., yeast diploid cells), such as through selecting preferred codons and/or altering the percentage AT through codon selection. It is known in the art that secretion efficiency and / or stability can be affected by the choice of secretion sequence and the optimal secretion sequence can vary between different proteins (see, e.g., Koganesawa et al., Protein Eng. 2001 Sep;14(9):705-10, which is incorporated by reference herein in its entirety). Many potentially suitable secretion signals are known in the art and can readily be tested for their effect upon yield and/or purity of a particular heterologous multi-subunit complex. Any secretion sequences may potentially be 39 2016273912 14 Dec 2016 used, including those present in secreted proteins of yeasts and other species, as well as engineered secretion sequences. Exemplary secretion sequences that may be utilized include: chicken lysozyme (CLY) signal peptide (MRSLLILVLCFLPLAALG (SEQ ID NO :414)), CLY-L8 (MRLLLLLLLLPLAALG (SEQ ID NO:415)), S. cerevisiae invertase (SUC2) signal peptide (MLLQAFLFLLAGFAAKISA (SEQ ID NO:416)), MF-alpha (Prepro) (MRFPSIFTAVLFAASSALA-APVNTTTE-EGVSLEKR (SEQ ID NO:417)), MF-alpha (Pre)-apv (MRFPSIFTAVLFAASSALA-APV (SEQ ID NO:418)), MF-alpha (Pre)-apv-SLEKR (MRFPSIFTAVLFAASSALA-APVSLEKR (SEQ ID NO:419)), MF-alpha (Prepro)-(EA)3 (MRFPSIFTAVLFAASSALA-APVNTTTE-EGVSLEKR-EAEAEA (SEQ ID NO:420)), aF signal peptide (MRFPSIFTAVLFAASSALA-APVNTTTE- DETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKE-EGVSLEKR (SEQ ID NO:421)), KILM1 signal peptide (MTKPTQVLVRSVSILFFITLLHLWALNDVAGPAETAPVSLLPR (SEQ ID NO :422)), repressible acid phosphatase (PHOl) signal peptide (MFSPILSLEIILALATLQSVFA (SEQ ID NO:423)), A. niger GOX signal peptide (MQTLLVSSLVVSLAAALPHYIR (SEQ ID NO:424)), Schwanniomyces occidentalis glucoamylase gene (GAM1) signal peptide (MIFLKLIKSIVIGLGLVSAIQA (SEQ ID NO:425)), human serum albumin (HSA) signal peptide with pro-sequence (MKWVTFISLLFLFSSAYSRGVFRR (SEQ ID NO:426)), human serum albumin (HSA) signal peptide without pro-sequence (MKWVTFISLLFLFSSAYS (SEQ ID NO:427)), ISN signal peptide (MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO:428)), IFN signal peptide (MKYTSYILAFQLCIVLGSLGCDLP (SEQ ID NO:429)), HGH signal peptide (MAADSQTPWLLTFSLLCLLWPQEPGA (SEQ ID NO:430)), phytohaemagglutinin (PHA) (MKKNRMMMMIWSVGVVWMLLLVGGSYG (SEQ ID NO:431)), Silkworm lysozyme (MQKLIIFALVVLCVGSEA (SEQ ID NO:432)), Human lysozyme (LYZ1) (MKALIVLGLVLLSVTVQG (SEQ ID NO:433)), activin receptor type-1 (MVDGVMILPVLIMIALPSPS (SEQ ID NO:434)), activin type II receptor (MGAAAKLAFAVFLISCSSG (SEQ ID NO:435)), P. pastoris immunoglobulin binding protein (PpBiP) (MLSLKPSWLTLAALMYAMLLVVVPFAKPVRA (SEQ ID NO:436)), and human antibody 3D6 light chain leader (MDMRVPAQLLGLLLLWLPGAKC (SEQ ID NO:437)). See Hashimoto et al., Protein Engineering vol. 11 no. 2 pp.75-77, 1998; Oka et al., Biosci Biotechnol Biochem. 1999 Nov; 63(11): 1977-83; Gellissen et al., FEMS Yeast Research 5 (2005) 1079-40 2016273912 14 Dec 2016 1096; Ma et al., Hepatology. 2005 Dec;42(6): 1355-63; Raemaekers et al., Eur J Biochem. 1999 Oct l;265(l):394-403; Koganesawa et al, Protein Eng. (2001) 14 (9): 705-710; Daly et al., Protein Expr Purif. 2006 Apr;46(2):456-67 ; Damasceno et al., Appl Microbiol Biotechnol (2007) 74:381-389; and Felgenhauer et al., Nucleic Acids Res. 1990 Aug 25; 18(16):4927, each of which is incorporated by reference herein in its entirety). The multi-subunit complex may also be secreted into the culture media without being operably linked or fused to a secretion signal. For example, it has been demonstrated that some heterologous polypeptides are secreted into the culture media when expressed in P. pastoris even without being linked or fused to a secretion signal. Additionally, the multi-subunit complex may be purified from host cells (which, for example, may be preferable if the complex is poorly secreted) using methods known in the art.

[00202] Media or cells comprising a desired multi-subunit complex may be recovered from the culture. Optionally, the secreted proteins may be purified. For example, cells comprising a desired multi-subunit complex may be lysed using mechanical, chemical, enzymatic, and/or osmotic methods (e.g., freezing with liquid nitrogen, using a homogenizer, spheroplasting, sonication, agitation in the presence of glass beads, using detergents, etc.). The desired multi-subunit complex may be concentrated, filtered, dialyzed, etc., using methods known in the art. The desired multi-subunit complex may be purified based on, for example, its molecular mass (e.g., size exclusion chromatography), isoelectric point (e.g., isoelectric focusing), electrophoretic mobility (e.g., gel electrophoresis), hydrophobic interaction chromatography (e.g., HPLC), charge (e.g., ion exchange chromatography), affinity (e.g., in the case of an antibody, binding to protein A, protein G, and/or an epitope to which the desired antibody binds), and/or glycosylation state (e.g., detected by lectin binding affinity). Multiple purification steps may be performed to obtain the desired level of purity. In an exemplary embodiment, the desired multi-subunit complex may be comprise an immunoglobulin constant domain and may be purified using protein A or protein G affinity, size exclusion chromatography, and lack of binding to lectin (to remove glycosylated forms). Optionally the A protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may be added to inhibit proteolytic degradation during purification. 41 2016273912 14 Dec 2016 [00203] Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or alternatively via a PCR/recombination method familiar to those skilled in the art (Gateway® Technology; Invitrogen, Carlsbad Calif.). If such sites do not exist, the synthetic oligonucleotide adapters or linkers may be used in accordance with conventional practice. Desired nucleic acids (including nucleic acids comprising operably linked sequences) may also be produced by chemical synthesis.

[00204] Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequences to which they are operably linked. Such promoters fall into several classes: inducible, constitutive, and repressible promoters (that increase levels of transcription in response to absence of a repressor). Inducible promoters may initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.

[00205] The yeast promoter fragment may also serve as the site for homologous recombination and integration of the expression vector into the same site in the yeast genome; alternatively a selectable marker is used as the site for homologous recombination. Pichia transformation is described in Cregg et al. (1985) Mol. Cell. Biol. 5:3376-3385, which is incorporated by reference herein in its entirety.

[00206] Examples of suitable promoters from Pichia include the CUP1 (induced by the level of copper in the medium), tetracycline inducible promoters, thiamine inducible promoters, AOX1 promoter (Cregg et al. (1989) Mol. Cell. Biol. 9:1316-1323); ICL1 promoter (Menendez et al. (2003) Yeast 20(13):1097-108); glyceraldehyde-3-phosphate dehydrogenase promoter 42 2016273912 14 Dec 2016 (GAP) (Waterham et al. (1997) Gene 186(l):37-44); and FLD1 promoter (Shen et al. (1998) Gene 216(1):93-102). The GAP promoter is a strong constitutive promoter and the CUP1, AOX and FLD1 promoters are inducible. Each foregoing reference is incorporated by reference herein in its entirety.

[00207] Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PH03, PH05, Pyk, and chimeric promoters derived therefrom. Additionally, non-yeast promoters may be used in the invention such as mammalian, insect, plant, reptile, amphibian, viral, and avian promoters. Most typically the promoter will comprise a mammalian promoter (potentially endogenous to the expressed genes) or will comprise a yeast or viral promoter that provides for efficient transcription in yeast systems.

[00208] The polypeptides of interest may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the polypeptide coding sequence that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed through one of the standard pathways available within the host cell. The S. cerevisiae alpha factor pre-pro signal has proven effective in the secretion of a variety of recombinant proteins from P. pastoris. Other yeast signal sequences include the alpha mating factor signal sequence, the invertase signal sequence, and signal sequences derived from other secreted yeast polypeptides. Additionally, these signal peptide sequences may be engineered to provide for enhanced secretion in diploid yeast expression systems. Other secretion signals of interest also include mammalian signal sequences, which may be heterologous to the protein being secreted, or may be a native sequence for the protein being secreted. Signal sequences include pre-peptide sequences, and in some instances may include propeptide sequences. Many such signal sequences are known in the art, including the signal sequences found on immunoglobulin chains, e.g., K28 preprotoxin sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequences, human Ig heavy chain, human Ig light chain, and the like. For example, see Hashimoto et. al. Protein Eng 11(2) 75 (1998); and Kobayashi et. al. Therapeutic Apheresis 2(4) 257 (1998), each of which is incorporated by reference herein in its entirety. 43 2016273912 14 Dec 2016 [00209] Transcription may be increased by inserting a transcriptional activator sequence into the vector. These activators are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Transcriptional enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter.

[00210] Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from 3' to the translation termination codon, in untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA.

[00211] Construction of suitable vectors containing one or more of the above-listed components employs standard ligation techniques or PCR/recombination methods. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required or via recombination methods. For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform host cells, and successful transformants selected by antibiotic resistance (e.g. ampicillin or Zeocin) where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion and/or sequenced.

[00212] As an alternative to restriction and ligation of fragments, recombination methods based on att sites and recombination enzymes may be used to insert DNA sequences into a vector. Such methods are described, for example, by Landy (1989) Ann. Rev. Biochem. 58:913-949; and are known to those of skill in the art. Such methods utilize intermolecular DNA recombination that is mediated by a mixture of lambda and E. coli-encoded recombination proteins. Recombination occurs between specific attachment (att) sites on the interacting DNA molecules. For a description of att sites see Weisberg and Landy (1983) Site-Specific Recombination in Phage Lambda, in Lambda II, Weisberg, ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press), pp. 211-250. The DNA segments flanking the recombination sites are 44 2016273912 14 Dec 2016 switched, such that after recombination, the att sites are hybrid sequences comprised of sequences donated by each parental vector. The recombination can occur between DNAs of any topology. Each foregoing reference is incorporated by reference herein in its entirety.

[00213] Att sites may be introduced into a sequence of interest by ligating the sequence of interest into an appropriate vector; generating a PCR product containing att B sites through the use of specific primers; generating a cDNA library cloned into an appropriate vector containing att sites; and the like.

[00214] Monocistronic and polycistronic genes. A monocistronic gene encodes an RNA that contains the genetic information to translate only a single protein. A polycistronic gene encodes an mRNA that contains the genetic information to translate more than one protein. The proteins encoded in a polycistronic gene may have the same or different sequences or a combination thereof. Dicistronic or bicistronic refers to a polycistronic gene that encodes two proteins. Polycistronic genes optionally include one or more internal ribosome entry site (IRES) elements to facilitate cap-independent initiation of translation, which may be situated at a location that can drive translation of the downstream protein coding region independently of the 5'-cap structure bound to the 5' end of the mRNA molecule. Any known IRES sequence (e.g., viral, eukaryotic, or artificial in origin) may be used. For example, the cricket paralysis virus IRES sequence in the intergenic region (IGR) may be used, as described in Thompson et al. (2001) PNAS 98:12972-12977. Optionally, IRES function may be potentiated by genetic alteration, e.g., by causing constitutive expression of eIF2 kinase GCN2 or disrupting two initiator tRNA(met) genes disrupted (id.).

[00215] Folding, as used herein, refers to the three-dimensional structure of polypeptides and proteins, where interactions between amino acid residues act to stabilize the structure. While non-covalent interactions are important in determining structure, usually the proteins of interest will have intra- and/or intermolecular covalent disulfide bonds formed by two cysteine residues. For naturally occurring proteins and polypeptides or derivatives and variants thereof, the proper folding is typically the arrangement that results in optimal biological activity, and can conveniently be monitored by assays for activity, e.g. ligand binding, enzymatic activity, etc. 45 2016273912 14 Dec 2016 [00216] In some instances, for example where the desired product is of synthetic origin, assays based on biological activity will be less meaningful. The proper folding of such molecules may be determined on the basis of physical properties, energetic considerations, modeling studies, and the like.

[00217] The expression host may be further modified by the introduction of sequences encoding one or more enzymes that enhance folding and disulfide bond formation, i.e. foldases, chaperoning, etc. Such sequences may be constitutively or inducibly expressed in the yeast host cell, using vectors, markers, etc. as known in the art. Preferably the sequences, including transcriptional regulatory elements sufficient for the desired pattern of expression, are stably integrated in the yeast genome through a targeted methodology.

[00218] For example, the eukaryotic PDI is not only an efficient catalyst of protein cysteine oxidation and disulfide bond isomerization, but also exhibits chaperone activity. Coexpression of PDI can facilitate the production of active proteins having multiple disulfide bonds. Also of interest is the expression of BIP (immunoglobulin heavy chain binding protein); cyclophilin; and the like. In one embodiment of the invention, the multi-subunit complex may be expressed from a yeast strain produced by mating, wherein each of the haploid parental strains expresses a distinct folding enzyme, e.g. one strain may express BIP, and the other strain may express PDI or combinations thereof.

[00219] The terms "desired protein" or "target protein" are used interchangeably and refer generally to a heterologous multi-subunit protein such as an antibody (e.g., a humanized antibody) or a binding portion thereof described herein.

[00220] The term "antibody" includes any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be "antibodies." A preferred source for producing antibodies useful as starting material according to the invention is rabbits. Numerous antibody coding sequences have been described; and others 46 2016273912 14 Dec 2016 may be raised by methods well-known in the art. Examples thereof include chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single chain antibodies such as scFvs, camelbodies, nanobodies, IgNAR (single-chain antibodies derived from sharks), small-modular immunopharmaceuticals (SMIPs), and antibody fragments such as Fabs, Fab', F(ab')2 and the like. See Streltsov V A, et al., Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype, Protein Sci. 2005 November; 14(11):2901-9. Epub 2005 Sep. 30; Greenberg A S, et al., A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks, Nature. 1995 Mar. 9; 374(6518): 168-73; Nuttall S D, et al., Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries, Mol Immunol. 2001 August; 38(4):313-26; Hamers-Casterman C, et al., Naturally occurring antibodies devoid of light chains, Nature. 1993 Jun. 3; 363(6428):446-8; Gill D S, et al., Biopharmaceutical drug discovery using novel protein scaffolds, Curr Opin Biotechnol. 2006 December; 17(6):653-8. Epub 2006 Oct. 19. Each foregoing reference is incorporated by reference herein in its entirety.

[00221] For example, antibodies or antigen binding fragments may be produced by genetic engineering. In this technique, as with other methods, antibody-producing cells are sensitized to the desired antigen or immunogen. The messenger RNA isolated from antibody producing cells is used as a template to make cDNA using PCR amplification. A library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigen specificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors. A combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library. This results in a library of clones which co-express a heavy and light chain (resembling the Fab fragment or antigen binding fragment of an antibody molecule). The vectors that carry these genes are co-transfected into a host cell. When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self-assemble to produce active antibodies that can be detected by screening with the antigen or immunogen.

[00222] Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof. Variant polypeptides can include 47 2016273912 14 Dec 2016 amino acid (aa) substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain, catalytic amino acid residues, etc). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.

[00223] Chimeric antibodies may be made by recombinant means by combining the variable light and heavy chain regions (Vl and Vh), obtained from antibody producing cells of one species with the constant light and heavy chain regions from another. Typically chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Pat. No. 5,624,659, incorporated herein by reference in its entirety). It is further contemplated that the human constant regions of chimeric antibodies of the invention may be selected from IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgGlO, IgGll, IgG12, IgG13, IgGl4, IgGl5, IgGl6, IgGl7, IgGl8 or IgGl9 constant regions.

[00224] Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal-derived antibody. This is accomplished by carefully examining the sequence of the hyper-variable loops of the variable regions of the monoclonal antibody, and fitting them to the structure of the human antibody chains. Although facially complex, the process is straightforward in practice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully herein by reference. Methods of humanizing antibodies have been described previously in issued U.S. Patent No. 7935340, the disclosure of which is incorporated herein by reference in its entirety.

In some instances, a determination of whether additional rabbit framework residues are required 48 2016273912 14 Dec 2016 to maintain activity is necessary. In some instances the humanized antibodies still requires some critical rabbit framework residues to be retained to minimize loss of affinity or activity. In these cases, it is necessary to change single or multiple framework amino acids from human germline sequences back to the original rabbit amino acids in order to have desired activity. These changes are determined experimentally to identify which rabbit residues are necessary to preserve affinity and activity.

[00225] In addition to entire immunoglobulins (or their recombinant counterparts), immunoglobulin fragments comprising the epitope binding site (e.g., Fab', F(ab')2, or other fragments) may be synthesized. "Fragment," or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques. For instance "Fv" immunoglobulins for use in the present invention may be produced by synthesizing a fused variable light chain region and a variable heavy chain region. Combinations of antibodies are also of interest, e.g. diabodies, which comprise two distinct Fv specificities. In another embodiment of the invention, SMIPs (small molecule immunopharmaceuticals), camelbodies, nanobodies, and IgNAR are encompassed by immunoglobulin fragments.

[00226] Immunoglobulins and fragments thereof may be modified post-translationally, e.g. to add effector moieties such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, toxins, substrates, bioluminescent materials, radioactive materials, chemiluminescent moieties and the like, or specific binding moieties, such as streptavidin, avidin, or biotin, and the like may be utilized in the methods and compositions of the present invention. Examples of additional effector molecules are provided infra.

[00227] Product-associated variant: a product other than the desired product (e.g., the desired multi-subunit complex) which is present in a preparation of the desired product and related to the desired product. Exemplary product-associated variants include truncated or elongated peptides, products having different glycosylation than the desired glycosylation (e.g., if an aglycosylated product is desired then any glycosylated product would be considered to be a product-associated variant), complexes having abnormal stoichiometry, improper assembly, abnormal disulfide linkages, abnormal or incomplete folding, aggregation, protease cleavage, or other abnormalities. Exemplary product-associated variants may exhibit alterations in one or 49 2016273912 14 Dec 2016 more of molecular mass (e.g., detected by size exclusion chromatography), isoelectric point (e.g., detected by isoelectric focusing), electrophoretic mobility (e.g., detected by gel electrophoresis), phosphorylation state (e.g., detected by mass spectrometry), charge to mass ratio (e.g., detected by mass spectrometry), mass or identity of proteolytic fragments (e.g., detected by mass spectrometry or gel electrophoresis), hydrophobicity (e.g., detected by HPLC), charge (e.g., detected by ion exchange chromatography), affinity (e.g., in the case of an antibody, detected by binding to protein A, protein G, and/or an epitope to which the desired antibody binds), and glycosylation state (e.g., detected by lectin binding affinity). Where the desired protein is an antibody, the term product-associate variant may include a glyco-heavy variant and/or half antibody species (described below).

[00228] Exemplary product-associated variants include variant forms that contain aberrant disulfide bonds. For example, most IgGl antibody molecules are stabilized by a total of 16 intrachain and inter-chain disulfide bridges, which stabilize the folding of the IgG domains in both heavy and light chains, while the inter-chain disulfide bridges stabilize the association between heavy and light chains. Other antibody types likewise contain characteristic stabilizing intrachain and inter-chain disulfide bonds. Further, some antibodies (including Ab-A and Ab-B disclosed herein) contain additional disulfide bonds referred to as non-canonical disulfide bonds. Thus, aberrant inter-chain disulfide bonds may result in abnormal complex stoichiometry, due to the absence of a stabilizing covalent linkage, and/or disulfide linkages to additional subunits. Additionally, aberrant disulfide bonds (whether inter-chain or intra-chain) may decrease structural stability of the antibody, which may result in decreased activity, decreased stability, increased propensity to form aggregates, and/or increased immunogenicity. Product-associated variants containing aberrant disulfide bonds may be detected in a variety of ways, including non-reduced denaturing SDS-PAGE, capillary electrophoresis, cIEX, mass spectrometry (optionally with chemical modification to produce a mass shift in free cysteines), size exclusion chromatography, HPLC, changes in light scattering, and any other suitable methods known in the art. See, e.g., The Protein Protocols Handbook 2002, Part V, 581-583, DOI: 10.1385/1 -59259-169-8:581; [00229] Half antibody, half-antibody species, or H1L1 refer to a protein complex that includes a single heavy and single light antibody chain, but lacks a covalent linkage to a second 50 2016273912 14 Dec 2016 heavy and light antibody chain. Two half antibodies may remain non-covalently associated under some conditions (which may give behavior similar to a full antibody, e.g., apparent molecular weight determined by size exclusion chromatography). Similarly, H2L1 refers to a protein complex that includes two heavy antibody chains and single light antibody chain, but lacks a covalent linkage to a second light antibody chain; these complexes may also non-covalently associate with another light antibody chain (and likewise give similar behavior to a full antibody). Like full antibodies, half antibody species and H2L1 species can dissociate under reducing conditions into individual heavy and light chains. Half antibody species and H2L1 species can be detected on a non-reduced SDS-PAGE gel as a species migrating at a lower apparent molecular weight than the full antibody, e.g., H1L1 migrates at approximately half the apparent molecular weight of the full antibody (e.g., about 75 kDa).

[00230] Glyco-heavy variant refers to a glycosylated product-associated variant sometimes present in antibody preparations and which contains at least a partial Fc sequence. The glyco-heavy variant is characterized by decreased electrophoretic mobility observable by SDS-PAGE (relative to a normal heavy chain), lectin binding affinity, binding to an anti-Fc antibody, and apparent higher molecular weight of antibody complexes containing the glyco-heavy variant as determined by size exclusion chromatography. See U.S. Provisional Application Ser. No. 61/525,307 (Atty. Docket No. 67858.730200), filed August 31, 2011 which is incorporated by reference herein in its entirety.

[00231] The term "polyploid yeast that stably expresses or expresses a desired secreted heterologous polypeptide for prolonged time" refers to a yeast culture that secretes said polypeptide for at least several days to a week, more preferably at least a month, still more preferably at least 1-6 months, and even more preferably for more than a year at threshold expression levels, typically at least 50-500 mg/liter (after about 90 hours in culture) and preferably substantially greater.

[00232] The term "polyploidal yeast culture that secretes desired amounts of recombinant polypeptide" refers to cultures that stably or for prolonged periods secrete at least at least 50-500 mg/liter, and most preferably 500-1000 mg/liter or more. 51 2016273912 14 Dec 2016 [00233] A polynucleotide sequence "corresponds" to a polypeptide sequence if translation of the polynucleotide sequence in accordance with the genetic code yields the polypeptide sequence (i.e., the polynucleotide sequence "encodes" the polypeptide sequence), one polynucleotide sequence "corresponds" to another polynucleotide sequence if the two sequences encode the same polypeptide sequence.

[00234] A "heterologous" region or domain of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

[00235] A "coding sequence" is an in-frame sequence of codons that (in view of the genetic code) correspond to or encode a protein or peptide sequence. Two coding sequences correspond to each other if the sequences or their complementary sequences encode the same amino acid sequences. A coding sequence in association with appropriate regulatory sequences may be transcribed and translated into a polypeptide. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Promoter sequences typically contain additional sites for binding of regulatory molecules (e.g., transcription factors) which affect the transcription of the coding sequence. A coding sequence is "under the control" of the promoter sequence or "operatively linked" to the promoter when RNA polymerase binds the promoter sequence in a cell and transcribes the coding sequence into mRNA, which is then in turn translated into the protein encoded by the coding sequence.

[00236] Vectors are used to introduce a foreign substance, such as DNA, RNA or protein, into an organism or host cell. Typical vectors include recombinant viruses (for polynucleotides) 52 2016273912 14 Dec 2016 and liposomes (for polypeptides). A "DNA vector" is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment. An "expression vector" is a DNA vector which contains regulatory sequences which will direct polypeptide synthesis by an appropriate host cell. This usually means a promoter to bind RNA polymerase and initiate transcription of mRNA, as well as ribosome binding sites and initiation signals to direct translation of the mRNA into a polypeptide(s). Incorporation of a polynucleotide sequence into an expression vector at the proper site and in correct reading frame, followed by transformation of an appropriate host cell by the vector, enables the production of a polypeptide encoded by said polynucleotide sequence.

[00237] "Amplification" of polynucleotide sequences is the in vitro production of multiple copies of a particular nucleic acid sequence. The amplified sequence is usually in the form of DNA. A variety of techniques for carrying out such amplification are described in the following review articles, each of which is incorporated by reference herein in its entirety: Van Brunt 1990, Bio/Technol., 8(4):291-294; and Gill and Ghaemi, Nucleosides Nucleotides Nucleic Acids. 2008 Mar;27(3):224-43. Polymerase chain reaction or PCR is a prototype of nucleic acid amplification, and use of PCR herein should be considered exemplary of other suitable amplification techniques.

[00238] The general structure of antibodies in most vertebrates (including mammals) is now well understood (Edelman, G. M., Ann. N.Y. Acad. Sci., 190: 5 (1971)). Conventional antibodies consist of two identical light polypeptide chains of molecular weight approximately 23.000 daltons (the "light chain"), and two identical heavy chains of molecular weight 53,000- 70.000 (the "heavy chain"). The four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" configuration. The "branch" portion of the "Y" configuration is designated the Fab region; the stem portion of the "Y" configuration is designated the Fc region. The amino acid sequence orientation runs from the N-terminal end at the top of the "Y" configuration to the C-terminal end at the bottom of each chain. The N-terminal end possesses the variable region having specificity for the antigen that elicited it, and is approximately 100 amino acids in length, there being slight variations between light and heavy chain and from antibody to antibody. 53 2016273912 14 Dec 2016 [00239] The variable region is linked in each chain to a constant region that extends the remaining length of the chain and that within a particular class of antibody does not vary with the specificity of the antibody (i.e., the antigen eliciting it). There are five known major classes of constant regions that determine the class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE corresponding to gamma, mu, alpha, delta, and epsilon heavy chain constant regions). The constant region or class determines subsequent effector function of the antibody, including activation of complement (Kabat, E. A., Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436, Holt, Rinehart, Winston (1976)), and other cellular responses (Andrews, D. W., et al., Clinical Immunobiology, pp 1-18, W. B. Sanders (1980);

Kohl, S., et al., Immunology, 48: 187 (1983)); while the variable region determines the antigen with which it will react. Light chains are classified as either kappa or lambda. Each heavy chain class can be paired with either kappa or lambda light chain. The light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages when the immunoglobulins are generated either by hybridomas or by B cells.

[00240] The expression "variable region" or "VR" refers to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

[00241] The expressions "complementarity determining region," "hypervariable region," or "CDR" refer to one or more of the hyper-variable or complementarity determining regions (CDRs) found in the variable regions of light or heavy chains of an antibody (See Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include the hypervariable regions as defined by Kabat et al. ("Sequences of Proteins of Immunological Interest," Kabat E., et al., US Dept, of Health and Human Services, 1983) or the hypervariable loops in 3-dimensional structures of antibodies (Chothia and Lesk, J Mol. Biol. 196 901-917 (1987)). The CDRs in each chain are held in close 54 2016273912 14 Dec 2016 proximity by framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site. Within the CDRs there are select amino acids that have been described as the selectivity determining regions (SDRs) which represent the critical contact residues used by the CDR in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34 (2005)).

[00242] The expressions "framework region" or "FR" refer to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody (See Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.

[00243] Anti-NGF Antibodies and Binding Fragments Thereof Having Binding Activity for NGF

[00244] Antibody Abl [00245] The invention contemplates methods of treating pain and the specific pain associated disorders using antibody Abl or fragments thereof, or an antibody or antibody fragment that binds to the same or overlapping epitope as Abl, for example as set forth below, alone or is association with another active agent, e.g., anNSAID or opioid analgesic, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75 and/or inhibits or prevents pain. In one embodiment, the invention includes chimeric antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AL VMTQTPS S V S AAV GGT VTINCQ ASQNIY SNLAWYQQRPGQRPKLLIY G ASNLDAGV PSRFRGSGSGTEYTLTISDLECDDVGTYYCQSAFDSDSTENTFGGGTEVVVKR (SEQ ID NO: 1).

[00246] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or 55 2016273912 14 Dec 2016 opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AL VMT QTPS S V S AAV GGT VTINCQ ASQNIY SNL A WY QQRPGQRPKLLIY G ASNLD AGV PSRFRGSGSGTEYTLTISDLECDDVGTYYCQSAFDSDSTENTFGGGTEVVVKRTVAAPSV FIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[00247] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: [00248] QSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGVI TSIGSTVYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGYDDYDEMTYFNIWGQ GTLVTVSS (SEQ ID NO: 3).

[00249] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: [00250] QSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGVI TSIGSTVYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGYDDYDEMTYFNIWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4).

[00251] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or 56 2016273912 14 Dec 2016 opioid analgesic, wherein the antibodies comprise one or more of the polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2, and/or one or more of the polypeptide sequences of SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00252] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibody is a fragment having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[00253] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2.

[00254] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include fragments having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10 which correspond to the complementarity-determining 57 2016273912 14 Dec 2016 regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4.

[00255] The invention also optionally contemplates antibody fragments which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 1; the variable heavy chain region of SEQ ID NO: 3; the complementarity-determining regions (SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7) of the variable light chain region of SEQ ID NO: 1; and the complementarity-determining regions (SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10) of the variable heavy chain region of SEQ ID NO: 3.

[00256] In a particularly preferred optional embodiment of the invention, the chimeric anti-NGF antibody is Abl, comprising, or alternatively consisting of, SEQ ID NO: 2 and SEQ ID NO: 4, and having at least one of the biological activities set forth herein.

[00257] In a further particularly preferred optional embodiment of the invention, antibody fragments for use herein comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Abl. With respect to antibody Abl, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 1 and the variable heavy chain sequence of SEQ ID NO: 3. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 1 and/or SEQ ID NO: 3 in said Fab while retaining binding specificity for NGF.

[00258] In one optional embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl. In another embodiment of the invention, anti-NGF antibodies such as Abl or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris. 58 2016273912 14 Dec 2016 [00259] Antibody Ab2 [00260] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric or humanized antibodies having binding specificity to NGF wherein the antibody is antibody Ab2 or fragments thereof, or an antibody or antibody fragment that binds to the same or overlapping epitope as Ab2, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQASQNIYSNLAWYQQKPGKAPKLLIYGASNLDAGVP SRFSGSGSGTEYTLTISSLQPDDFATYYCQSAFDSDSTENTFGGGTKVEIKR (SEQ ID NO: [00261] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQASQNIYSNLAWYQQKPGKAPKLLIYGASNLDAGVP SRFSGSGSGTEYTLTISSLQPDDFATYYCQSAFDSDSTENTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[00262] The invention further optionally includes chimeric or humanized antibodies having binding specificity to NGF for treatment or prevention of pain and pain associated conditions and possessing a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSSYAMSWVRQAPGKGLEWVGVITSIGSTV YASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYDDYDEMTYFNIWGQGT LVTVSS (SEQ ID NO: 13).

[00263] The invention also optionally includes chimeric or humanized antibodies having binding specificity to NGF for treatment or prevention of pain and pain associated conditions and 59 2016273912 14 Dec 2016 possessing a heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSSYAMSWVRQAPGKGLEWVGVITSIGSTV Y AS S AKGRFTISRDN SKNTL YLQMN SLRAEDTAVYY C ARGYDD YDEMTYFNIWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYASTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14).

[00264] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12, and/or one or more of the polypeptide sequences of SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00265] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[00266] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions 60 2016273912 14 Dec 2016 optionally comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12.

[00267] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14.

[00268] The invention also optionally contemplates antibody fragments which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 11; the variable heavy chain region of SEQ ID NO: 13; the complementarity-determining regions (SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17) of the variable light chain region of SEQ ID NO: 11; and the complementarity-determining regions (SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20) of the variable heavy chain region of SEQ ID NO: 13.

[00269] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions optionally is Ab2, comprising, or alternatively consisting of, SEQ ID NO: 12 and SEQ ID NO: 14, and having at least one of the biological activities set forth herein.

[00270] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF or a Fab or other monovalent antibody fragment that binds to the same or overlapping epitope as Ab2,. With respect to antibody Ab2, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 11 and the variable heavy chain sequence of SEQ ID NO: 13. 61 2016273912 14 Dec 2016

This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 11 and/or SEQ ID NO: 13 in said Fab while retaining binding specificity for NGF.

[00271] In one optional embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab2. In another embodiment of the invention, anti-NGF antibodies such as Ab2 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00272] Antibody Ab 3 [00273] The invention contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric or humanized antibodies having binding specificity to NGF wherein the antibody is Ab3 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Ab3, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75. In one embodiment, the invention includes chimeric antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AVFTQTPSPVSAAMGDTVTIKCQSSQSVYKNNYFSWYQQKPGQPPRLFIYDASNLPSGV PSRFSGSGSGTQFTFTISGVQCDDAATYYCFGDYDDDADNAFGGGTEVWKR (SEQ ID NO: 21).

[00274] The invention also includes chimeric antibodies or treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AVFTQTPSPVSAAMGDTVTIKCQSSQSVYKNNYFSWYQQKPGQPPRLFIYDASNFPSGV PSRFSGSGSGTQFTFTISGVQCDDAATYYCFGDYDDDADNAFGGGTEVVVKRTVAAPS VFIFPPSDEQFKSGTASVVCFFNNFYPREAKVQWKVDNAFQSGNSQESVTEQDSKDSTY SESSTETESKADYEKHKVYACEVTHQGESSPVTKSFNRGEC (SEQ ID NO: 22). 62 2016273912 14 Dec 2016 [00275] The invention further includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYVMIWVRQAPGKGLEYIGITWSAGTYYAS WAKGRFTISKTSSTTVDLKITSPTTEDTATYFCAGGGGSIYDIWGPGTLVTVSS (SEQ ID NO: 23).

[00276] The invention also includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: Q S VEES GGRL VTPGTPLTLT CT V S GF SLS S Y VMIWVRQ APGKGLEYIGIT W S AGT YYAS WAKGRFTISKTSSTTVDLKITSPTTEDTATYFCAGGGGSIYDIWGPGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPE VT C V V VD V SHEDPE VKFNWYVDGVE VHNAKTKPREEQ YAST YRV V S VLT VLHQD WLN GKE YKCKV SNKALP APIEKTISKAKGQPREPQ VYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24).

[00277] The invention further contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22, and/or one or more of the polypeptide sequences of SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them. 63 2016273912 14 Dec 2016 [00278] The invention also contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 21 or SEQ ID NO: 22. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 23 or SEQ ID NO: 24.

[00279] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22.

[00280] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24.

[00281] The invention also optionally contemplates antibody fragments which include one or more of the antibody fragments described herein for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 21; the variable heavy chain region of SEQ ID NO: 23; the complementarity-determining regions (SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27) of the variable light chain region of SEQ ID NO: 21; and the complementarity-determining regions (SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30) of the variable heavy chain region of SEQ ID NO: 23.

[00282] In a particularly preferred optional embodiment of the invention, the chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab3, 64 2016273912 14 Dec 2016 comprising, or alternatively consisting of, SEQ ID NO: 22 and SEQ ID NO: 24, and having at least one of the biological activities set forth herein.

[00283] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Ab3. With respect to antibody Ab3, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 21 and the variable heavy chain sequence of SEQ ID NO: 23. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 21 and/or SEQ ID NO: 23 in said Fab while retaining binding specificity for NGF.

[00284] In one optional embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab3. In another embodiment of the invention, anti-NGF antibodies such as Ab3 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00285] Antibody Ab4 [00286] The invention contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric or humanized antibodies having binding specificity to NGF wherein the antibody is antibody Ab4 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Ab4, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75 and/or for preventing or effectively treating pain. In one embodiment, the invention includes humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: 65 2016273912 14 Dec 2016 [00287] DIQMTQSPSTLSASVGDRVTITCQSSQSVYKNNYLSWYQQKPGKAPKLLI YDASNLPSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCLGDYDDDADNAFGGGTKVEI KR (SEQ ID NO: 31).

[00288] The invention also includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQSSQSVYKNNYLSWYQQKPGKAPKLLIYDASNLPSG VPSRFSGSGSGTEFTLTISSLQPDDFATYYCLGDYDDDADNAFGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 32).

[00289] The invention further includes humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSSYVMIWVRQAPGKGLEYIGITWSAGTYY ASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGGGSIYDIWGQGTLVTVSS (SEQ ID NO: 33).

[00290] The invention also includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSSYVMIWVRQAPGKGLEYIGITWSAGTYY ASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGGGSIYDIWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34).

[00291] The invention further contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID 66 2016273912 14 Dec 2016 NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32, and/or one or more of the polypeptide sequences of SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00292] The invention also contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 31 or SEQ ID NO: 32. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[00293] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32.

[00294] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34. 67 2016273912 14 Dec 2016 [00295] The invention also optional contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 31; the variable heavy chain region of SEQ ID NO: 33; the complementarity-determining regions (SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37) of the variable light chain region of SEQ ID NO: 31; and the complementarity-determining regions (SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40) of the variable heavy chain region of SEQ ID NO: 33.

[00296] In a particularly preferred embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab4, comprising, or alternatively consisting of, SEQ ID NO: 32 and SEQ ID NO: 34, and having at least one of the biological activities set forth herein.

[00297] In a further particularly preferred embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Abl4. With respect to antibody Ab4, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 31 and the variable heavy chain sequence of SEQ ID NO: 33. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 31 and/or SEQ ID NO: 33 in said Fab while retaining binding specificity for NGF.

[00298] In one embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab4. In another embodiment of the invention, anti-NGF antibodies such as Ab4 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00299] Antibody Ab5 68 2016273912 14 Dec 2016 [00300] The invention contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies optionally include Ab5 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Ab5, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AYDMTQTPASVEVAVGGTVTIKCQASQSIYSNLAWYQQRPGQPPKLLIYDASTLESGVP SRFKGSGSGTEYTLTISGVECADAASYYCQQGFTVSDIDNAFGGGTEVVVKR (SEQ ID NO: 41).

[00301] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AYDMTQTPASVEVAVGGTVTIKCQASQSIYSNLAWYQQRPGQPPKLLIYDASTLESGVP SRFKGSGSGTEYTLTISGVECADAASYYCQQGFTVSDIDNAFGGGTEVWKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42).

[00302] The invention further optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: Q S VEES GGRL VTPGTPLTLT CT V S GF SLSNYAV GWVRQ APGKGLE WIGIIGRNGNTWY A SWARGRFTISKTSTTVDLKITSPTSEDTATYFCARGYGRSVAYYVFNIWGPGTLVTVSS (SEQ ID NO: 43).

[00303] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below:

Q S VEES GGRL VTPGTPLTLT CT V S GF SLSN Y AV GWVRQ APGKGLE WIGIIGRNGNTWY A SWARGRFTISKTSTTVDLKITSPTSEDTATYFCARGYGRSVAYYVFNIWGPGTLVTVSSA 69 2016273912 14 Dec 2016

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP

SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44).

[00304] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42, and/or one or more of the polypeptide sequences of SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof optionally comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00305] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 41 or SEQ ID NO: 42. In another optional embodiment of the invention, antibody fragments of the invention for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 43 or SEQ ID NO: 44.

[00306] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47 which correspond to the 70 2016273912 14 Dec 2016 complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42.

[00307] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions optionally comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.

[00308] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 41; the variable heavy chain region of SEQ ID NO: 43; the complementarity-determining regions (SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47) of the variable light chain region of SEQ ID NO: 41; and the complementarity-determining regions (SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50) of the variable heavy chain region of SEQ ID NO: 43.

[00309] In a particularly preferred optional embodiment of the invention, the optionally included chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab5, comprising, or alternatively consisting of, SEQ ID NO: 42 and SEQ ID NO: 44, and having at least one of the biological activities set forth herein.

[00310] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF or another Fab or antibody fragment that binds to the same or overlapping epitope as Ab5. With respect to antibody Ab5, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 41 and the variable heavy chain sequence of SEQ ID NO: 43. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 41 and/or SEQ ID NO: 43 in said Fab while retaining binding specificity for NGF. 71 2016273912 14 Dec 2016 [00311] In one embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab5. In another embodiment of the invention, anti-NGF antibodies such as Ab5 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00312] Antibody Ab6 [00313] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include Ab6 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Ab6, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKR (SEQ ID NO: [00314] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 52).

[00315] The invention further optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: 72 2016273912 14 Dec 2016 E VQL VE S GGGL V QPGGSLRLS C AAS GFT V SN Y AV G WVRQ APGKGLE WV GIIGRN GNT WY AS S ARGRFTISRDNSKNTLYLQMNSLRAEDT AVYY C ARGY GRS V AYYVFNIWGPGT LVTVSS (SEQ ID NO: 53).

[00316] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT WY AS S ARGRFTISRDNSKNTLYLQMNSLRAEDT AVYY CARGY GRSV AYYVFNI WGPGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYASTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54).

[00317] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52, and/or one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00318] The invention also optionally contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention comprise, or 73 2016273912 14 Dec 2016 alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ ID NO: 52. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

[00319] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52.

[00320] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54.

[00321] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 51; the variable heavy chain region of SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60) of the variable heavy chain region of SEQ ID NO: 53.

[00322] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab6, comprising, or alternatively consisting of, SEQ ID NO: 52 and SEQ ID NO: 54, and having at least one of the biological activities set forth herein. 74 2016273912 14 Dec 2016 [00323] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Ab6. With respect to antibody Ab6, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 51 and the variable heavy chain sequence of SEQ ID NO: 53. This optional embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 51 and/or SEQ ID NO: 53 in said Fab while retaining binding specificity for NGF.

[00324] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab6. In another embodiment of the invention, anti-NGF antibodies such as Ab6 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00325] Antibody Ab7 [00326] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include Ab7 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Ab7, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: ADVVMTQTPAS VSQPVGGTVTIKCQ ASEDIYNLLAWY QQKPGQPPKLLIY S ASTL ASGV PSRFKGSGSGTEYTLTISGLECADAATYYCQNNYLVTTYGVAFGGGTEVWKR (SEQ ID NO: 61). 75 2016273912 14 Dec 2016 [00327] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: ADVVMTQTPASVSQPVGGTVTIKCQASEDIYNLLAWYQQKPGQPPKLLIYSASTLASGV PSRFKGSGSGTEYTLTISGLECADAATYYCQNNYLVTTYGVAFGGGTEWVKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 62).

[00328] The invention further optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QEQLKESGGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEYIGYIDTDTSAYYA SWVKGRFTISRTSTTVDLKITSPTTEDTATYFCARSYAAYGGYPATFDPWGPGTLVTVSS (SEQ ID NO: 63).

[00329] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QEQLKE S GGRL VTPGTPLTLT CT V S GF SL S S Y AMI WVRQ APGKGLE YIGYIDTDTS A Y Y A S WVKGRFTISRTSTTVDLKITSPTTEDTATYF C ARSY AAY GGYP ATFDPWGPGTLVTV SS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL Y SL S SWT VP S S SLGT QT YICN VNHKP SNTKVDKRVEPKSCDKTHT CPPCP APELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY ASTYRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 64).

[00330] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62, and/or one or 76 2016273912 14 Dec 2016 more of the polypeptide sequences of SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00331] The invention also optionally contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 61 or SEQ ID NO: 62. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 63 or SEQ ID NO: 64.

[00332] In a further optional embodiment of the invention, fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62.

[00333] In a further optional embodiment of the invention, fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64.

[00334] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, 77 2016273912 14 Dec 2016 including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 61; the variable heavy chain region of SEQ ID NO: 63; the complementarity-determining regions (SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67) of the variable light chain region of SEQ ID NO: 61; and the complementarity-determining regions (SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70) of the variable heavy chain region of SEQ ID NO: 63.

[00335] In a particularly preferred optional embodiment of the invention, the chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab7, comprising, or alternatively consisting of, SEQ ID NO: 62 and SEQ ID NO: 64, and having at least one of the biological activities set forth herein.

[00336] In a further particularly optionally preferred embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Ab7. With respect to antibody Ab7, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 61 and the variable heavy chain sequence of SEQ ID NO: 63. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 61 and/or SEQ ID NO: 63 in said Fab while retaining binding specificity for NGF.

[00337] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab7. In another embodiment of the invention, anti-NGF antibodies such as Ab7 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00338] Antibody Ab8 [00339] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include Ab8 or fragments thereof, or another antibody 78 2016273912 14 Dec 2016 or fragment that binds to the same or overlapping epitope as Ab8, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: DIQMTQSPSSLSASVGDRVTITCQASEDIYNLLAWYQQKPGKVPKLLIYSASTLASGVPS RFSGSGSGTDYTLTISSLQPEDVATYYCQNNYLVTTYGVAFGGGTKVEIKR (SEQ ID NO: 71).

[00340] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: DIQMTQSPSSLSASVGDRVTITCQASEDIYNLLAWYQQKPGKVPKLLIYSASTLASGVPS RFSGSGSGTDYTLTISSLQPEDVATYYCQNNYLVTTYGVAFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 72).

[00341] The invention further optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: Q V QL VES GGG V V QPGRSLRL S C AAS GFTF S S Y AMI WVRQ APGKGLE YIGYIDTDTS AYY AS S VKGRFTISRDNSKNTL YLQMS SLRAEDT AVYY C ARS YAAY GGYPATFDPWGQGTL VTVSS (SEQ ID NO: 73).

[00342] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below:

Q V QL VES GGG V V QPGRSLRL S C AAS GFTF S S Y AMI WVRQ APGKGLE YIGYIDTDTS AYY AS S VKGRFTISRDNSKNTL YLQMS SLRAEDT AVYY C ARS Y AAY GGYPATFDPWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP 79 2016273912 14 Dec 2016 REEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK FTVDKSRWQQGNVFSCSVMHEAFHNHYTQKSFSFSPGK (SEQ ID NO: 74).

[00343] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72, and/or one or more of the polypeptide sequences of SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00344] The invention also optionally contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 73 or SEQ ID NO: 74.

[00345] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72.

[00346] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated 80 2016273912 14 Dec 2016 conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74.

[00347] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 71; the variable heavy chain region of SEQ ID NO: 73; the complementarity-determining regions (SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77) of the variable light chain region of SEQ ID NO: 71; and the complementarity-determining regions (SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80) of the variable heavy chain region of SEQ ID NO: 73.

[00348] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab8, comprising, or alternatively consisting of, SEQ ID NO: 72 and SEQ ID NO: 74, and having at least one of the biological activities set forth herein.

[00349] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab8, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 71 and the variable heavy chain sequence of SEQ ID NO: 73 or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Ab8. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 71 and/or SEQ ID NO: 73 in said Fab while retaining binding specificity for NGF.

[00350] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab8. In another embodiment of the invention, anti-NGF antibodies such as Ab8 or Fab fragments thereof may be produced via expression in mammalian 81 2016273912 14 Dec 2016 cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00351] Antibody Ab9 [00352] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include Ab9 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Ab9, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one optional embodiment, the invention includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AYDMTQTPASVSAAVGGTVTIKCQASENIGSYLAWYQQKPGQPPELLIYRASTLASGVP SRFKGSGSGTQFTLTISGVECADAATYYCQQGYNSENLDNAFGGGTEVVVKR (SEQ ID NO: 81).

[00353] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AYDMTQTPASVSAAVGGTVTIKCQASENIGSYLAWYQQKPGQPPELLIYRASTLASGVP SRFKGSGSGTQFTLTISGVECADAATYYCQQGYNSENLDNAFGGGTEVVVKRTVAAPS VFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 82).

[00354] The invention further optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QSVEESGGRLVTPGTPLTLTCTVSGIDLSMYSMGWVRQAPGKGLEYIGWISYGGTAYYA SWAKGRFTISKTSTTVELKITSPTIEDTATYFCARETPVNYYLDIWGQGTLVTVSS (SEQ ID NO: 83). 82 2016273912 14 Dec 2016 [00355] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QSVEESGGRLVTPGTPLTLTCTVSGIDLSMYSMGWVRQAPGKGLEYIGWISYGGTAYYA SWAKGRFTISKTSTTVELKITSPTIEDTATYFCARETPVNYYLDIWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RW SVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84).

[00356] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82, and/or one or more of the polypeptide sequences of SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00357] The invention also optionally contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 81 or SEQ ID NO: 82. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 83 or SEQ ID NO: 84. 83 2016273912 14 Dec 2016 [00358] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82.

[00359] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84.

[00360] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 81; the variable heavy chain region of SEQ ID NO: 83; the complementarity-determining regions (SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87) of the variable light chain region of SEQ ID NO: 81; and the complementarity-determining regions (SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90) of the variable heavy chain region of SEQ ID NO: 83.

[00361] In a particularly preferred optional embodiment of the invention, the chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab9, comprising, or alternatively consisting of, SEQ ID NO: 82 and SEQ ID NO: 84, and having at least one of the biological activities set forth herein.

[00362] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab9, the Fab fragment includes the variable light chain sequence 84 2016273912 14 Dec 2016 of SEQ ID NO: 81 and the variable heavy chain sequence of SEQ ID NO: 83 or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Ab9. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 81 and/or SEQ ID NO: 83 in said Fab while retaining binding specificity for NGF.

[00363] In one embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab9. In another optional embodiment of the invention, anti-NGF antibodies such as Ab9 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00364] Antibody AblO

[00365] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include AblO or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as AblO, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AYDMTQSPSSLSASVGDRVTITCQASENIGSYLAWYQQKPGKVPKLLIYRASTLASGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQQGYNSENLDNAFGGGTKVEIKR (SEQ ID NO: 91).

[00366] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AYDMTQSPSSLSASVGDRVTITCQASENIGSYLAWYQQKPGKVPKLLIYRASTLASGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQQGYNSENLDNAFGGGTKVEIKRTVAAPSVFI 85 2016273912 14 Dec 2016 FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 92).

[00367] The invention further optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QVQLVESGGGVVQPGRSLRLSCAASGFTFSMYSMGWVRQAPGKGLEYIGWISYGGTAY YASSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARETPVNYYLDIWGQGTLVTV SS (SEQ ID NO: 93).

[00368] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QVQLVESGGGWQPGRSLRLSCAASGFTFSMYSMGWVRQAPGKGLEYIGWISYGGTAY YASSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARETPVNYYLDIWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSYYTYPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 94).

[00369] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92, and/or one or more of the polypeptide sequences of SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist 86 2016273912 14 Dec 2016 of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00370] The invention also optionally contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 91 or SEQ ID NO: 92. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 93 or SEQ ID NO: 94.

[00371] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions optionally comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92.

[00372] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.

[00373] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 91; the variable heavy chain region of SEQ ID NO: 93; the complementarity-determining regions (SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97) of the variable light chain region of SEQ ID NO: 91; and the 87 2016273912 14 Dec 2016 complementarity-determining regions (SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100) of the variable heavy chain region of SEQ ID NO: 93.

[00374] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is AblO, comprising, or alternatively consisting of, SEQ ID NO: 92 and SEQ ID NO: 94, and having at least one of the biological activities set forth herein.

[00375] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody AblO, the Fab fragment for treatment or prevention of pain and pain associated conditions includes the variable light chain sequence of SEQ ID NO: 91 and the variable heavy chain sequence of SEQ ID NO: 93 or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as AblO. This embodiment of the invention further optionally contemplates additions, deletions, and variants of SEQ ID NO: 91 and/or SEQ ID NO: 93 in said Fab while retaining binding specificity for NGF.

[00376] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of AblO. In another embodiment of the invention, anti-NGF antibodies such as AblO or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00377] Antibody Abl 1 [00378] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include Abl 1 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Abl 1, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with 88 2016273912 14 Dec 2016

TrkA and the association of NGF with p75. In one optional embodiment, the invention includes chimeric antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AFELTQTPSSVEAAVGGTVTIKCQASQNIVTNLAWYQQKPGQPPKLLIYGASTLASGVSS RFKGSGSGTQFTLTISDLECADAATYFCQSYDGFNSAGFGGGTEVVVKR (SEQ ID NO: 101).

[00379] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AFELTQTPSSVEAAVGGTVTIKCQASQNIVTNLAWYQQKPGQPPKLLIYGASTLASGVSS RFKGSGSGTQFTLTISDLECADAATYFCQSYDGFNSAGFGGGTEVVVKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 102).

[00380] The invention further optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGTPLTLTCTASGFSLSGYDMSWVRQAPGKGLEYIGLISYDGNTYYA TWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARSLYAGPNAGIGPFNIWGQGTLVTVSS (SEQ ID NO: 103).

[00381] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGTPLTLTCTASGFSLSGYDMSWVRQAPGKGLEYIGLISYDGNTYYA TWAKGRFTISKTSTTVDLKITSPTTEDTATYFC ARSLY AGPNAGIGPFNIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL Y SLS S V VTVPS S SLGT QT YICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCP APELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR 89 2016273912 14 Dec 2016 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 104).

[00382] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102, and/or one or more of the polypeptide sequences of SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof optionally comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00383] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 101 or SEQ ID NO: 102. In another optional embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 103 or SEQ ID NO: 104.

[00384] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102.

[00385] In a further optional embodiment of the invention, fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF 90 2016273912 14 Dec 2016 comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104.

[00386] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 101; the variable heavy chain region of SEQ ID NO: 103; the complementarity-determining regions (SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107) of the variable light chain region of SEQ ID NO: 101; and the complementarity-determining regions (SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110) of the variable heavy chain region of SEQ ID NO: 103.

[00387] In a particularly preferred optional embodiment of the invention, the chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl 1, comprising, or alternatively consisting of, SEQ ID NO: 102 and SEQ ID NO: 104, and having at least one of the biological activities set forth herein.

[00388] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl 1, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 101 and the variable heavy chain sequence of SEQ ID NO: 103 or comprises another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Abl 1. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 101 and/or SEQ ID NO: 103 in said Fab while retaining binding specificity for NGF.

[00389] In one optional embodiment of the invention described herein (infra), Fab fragments may for treatment or prevention of pain and pain associated conditions be produced by enzymatic digestion (e.g., papain) of Abl 1. In another optional embodiment of the invention, 91 2016273912 14 Dec 2016 anti-NGF antibodies such as Abl 1 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00390] Antibody Abl 2 [00391] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include Abl2 or fragments thereof, f or another antibody or antibody fragment that binds to the same or overlapping epitope as Abl2, or example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AFQMTQSPSSLSASVGDRVTITCQASQNIVTNLAWYQQKPGKYPKLLIYGASTLASGVP SRFSGSGSGTDFTLTISSLQPEDVATYYCQSYDGFNSAGFGGGTKVEIKR (SEQ ID NO: 111).

[00392] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AFQMTQSPSSLSASVGDRVTITCQASQNIVTNLAWYQQKPGKVPKLLIYGASTLASGVP SRFSGSGSGTDFTLTISSLQPEDVATYYCQSYDGFNSAGFGGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 112).

[00393] The invention further optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QVQLVESGGGVVQPGRSLRLSCAASGFSLSGYDMSWVRQAPGKGLEWVGLISYDGNT 92 2016273912 14 Dec 2016 YYATSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSLYAGPNAGIGPFNIWGQG TLVTVSS (SEQ ID NO: 113).

[00394] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QVQLVESGGGVVQPGRSLRLSCAASGFSLSGYDMSWVRQAPGKGLEWVGLISYDGNT YYATSAKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARSLYAGPNAGIGPFNIWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQ Y AST YRV V S VLT VLHQD WLN GKE YKCKV SNKALP APIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114).

[00395] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 115; SEQ ID NO: 116; and SEQ ID NO: 117 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 112, and/or one or more of the polypeptide sequences of SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00396] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one optional embodiment of the invention, antibody fragments of the invention comprise, or 93 2016273912 14 Dec 2016 alternatively consist of, the polypeptide sequence of SEQ ID NO: 111 or SEQ ID NO: 112. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 113 or SEQ ID NO: 114.

[00397] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 115; SEQ ID NO: 116; and SEQ ID NO: 117 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 112.

[00398] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114.

[00399] The invention also optionally contemplates antibody fragments which include one or more of the antibody fragments described herein. In one optional embodiment of the invention, fragments of the antibodies having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 111; the variable heavy chain region of SEQ ID NO: 113; the complementaritydetermining regions (SEQ ID NO: 115; SEQ ID NO: 116; and SEQ ID NO: 117) of the variable light chain region of SEQ ID NO: 111; and the complementarity-determining regions (SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120) of the variable heavy chain region of SEQ ID NO: 113.

[00400] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl2, comprising, or alternatively consisting of, SEQ ID NO: 112 and SEQ ID NO: 114, and having at least one of the biological activities set forth herein. 94 2016273912 14 Dec 2016 [00401] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl2, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 111 and the variable heavy chain sequence of SEQ ID NO: 113 or comprises another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Abl2. This optional embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 111 and/or SEQ ID NO: 113 in said Fab while retaining binding specificity for NGF.

[00402] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl2. In another embodiment of the invention, anti-NGF antibodies such as Abl2 or Fab fragments thereof for treatment or prevention of pain and pain associated conditions may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00403] Antibody Abl 3 [00404] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include Abl3 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Abl3, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below:

AAVETQTPSPVSAAVGGTVSISCQSSQNVYKNNYFSWYQQKPGQPPKFFIYKASTFASG 95 2016273912 14 Dec 2016 VPSRFKGGGSGTDFTLTISDVQCDAAATYYCAGGYTSSSDNAFGGGTEVVVKR (SEQ ID NO: 121).

[00405] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AAVLTQTPSPVSAAVGGTVSISCQSSQNVYKNNYLSWYQQKPGQPPKLLIYKASTLASG VPSRFKGGGSGTDFTLTISDVQCDAAATYYCAGGYTSSSDNAFGGGTEVVVKRTVAAP SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 122).

[00406] The invention further optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QSVEASGGRLVTPGTPLTLTCTASGFSLSTYWMSWVRQAPGKGLEWIGDIYFSNEETNY ASWAKGRFTISKTSTTVDLNVISPTTEDTATYFCARGSPDVDIGIDMWGPGTLVTVSS (SEQ ID NO: 123).

[00407] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QSVEASGGRLVTPGTPLTLTCTASGFSLSTYWMSWVRQAPGKGLEWIGDIYFSNEETNY ASWAKGRFTISKTSTTVDLNVISPTTEDTATYFCARGSPDVDIGIDMWGPGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVT C VWD V SHEDPE VKFN WYVDG VE VHNAKTKPREEQ Y A STYRW S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 124).

[00408] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127 which correspond to the 96 2016273912 14 Dec 2016 complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122, and/or one or more of the polypeptide sequences of SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00409] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 121 or SEQ ID NO: 122. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 123 or SEQ ID NO: 124.

[00410] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122.

[00411] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124. 97 2016273912 14 Dec 2016 [00412] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 121; the variable heavy chain region of SEQ ID NO: 123; the complementarity-determining regions (SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127) of the variable light chain region of SEQ ID NO: 121; and the complementarity-determining regions (SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130) of the variable heavy chain region of SEQ ID NO: 123.

[00413] In a particularly preferred optional embodiment of the invention, the chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl3, comprising, or alternatively consisting of, SEQ ID NO: 122 and SEQ ID NO: 124, and having at least one of the biological activities set forth herein.

[00414] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl3, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 121 and the variable heavy chain sequence of SEQ ID NO: 123 or comprises another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as Abl3. This optional embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 121 and/or SEQ ID NO: 123 in said Fab while retaining binding specificity for NGF.

[00415] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl3. In another embodiment of the invention, anti-NGF antibodies such as Abl3 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris. 98 2016273912 14 Dec 2016 [00416] Antibody Ab 14 [00417] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is Abl4 or fragments thereof, or another antibody or fragment that binds to the same or overlapping epitope as Abl4, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one optional embodiment, the invention includes chimeric or humanized antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: DIQMTQSPSSLSASVGDRVTITCQSSQNVYKNNYLSWYQQKPGKVPKLLIYKASTLASG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYTSSSDNAFGGGTKVEIKR (SEQ ID NO: 131).

[00418] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: DIQMTQSPSSLSASVGDRVTITCQSSQNVYKNNYLSWYQQKPGKVPKLLIYKASTLASG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYTSSSDNAFGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 132).

[00419] The invention further optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSTYWMSWVRQAPGKGLEWVGDIYFSNEET NYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSPDVDIGIDMWGPGTLV TVSS (SEQ ID NO: 133).

[00420] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: 99 2016273912 14 Dec 2016 EVQLVESGGGLVQPGGSLRLSCAASGFTVSTYWMSWVRQAPGKGLEWVGDIYFSNEET NY AS S AKGRFTISRDN SKNTL YLQMNSLRAEDT AVYY C ARGSPD VDIGIDM WGPGTL V TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 134).

[00421] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132, and/or one or more of the polypeptide sequences of SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00422] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 131 or SEQ ID NO: 132. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 133 or SEQ ID NO: 134.

[00423] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated 100 2016273912 14 Dec 2016 conditions optionally comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132.

[00424] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134.

[00425] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 131; the variable heavy chain region of SEQ ID NO: 133; the complementarity-determining regions (SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137) of the variable light chain region of SEQ ID NO: 131; and the complementarity-determining regions (SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140) of the variable heavy chain region of SEQ ID NO: 133.

[00426] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl4, comprising, or alternatively consisting of, SEQ ID NO: 132 and SEQ ID NO: 134, and having at least one of the biological activities set forth herein.

[00427] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl4, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 131 and the variable heavy chain sequence of SEQ ID NO: 133 or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as 101 2016273912 14 Dec 2016

Abl4. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 131 and/or SEQ ID NO: 133 in said Fab while retaining binding specificity for NGF.

[00428] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl4. In another embodiment of the invention, anti-NGF antibodies such as Abl4 or Fab fragments thereof for treatment or prevention of pain and pain associated conditions may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00429] Antibody Abl 5 [00430] The invention contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., anNSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is Abl5 or fragments thereof, for example as set forth below, or comprises another antibody or antibody fragment that binds to the same or overlapping epitope as Abl5, in a therapeutically effective amount which inhibits the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75. In one embodiment, the invention includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AAVLTQTPSPVSAAVGDTVTIKCQSSQSVYKNNYLSWYQQKPGQPPKLLIYDASNLPSG VPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGDYDDDTDNGFGGGTEVVVKR (SEQ ID NO: 141).

[00431] The invention also includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below:

AAVLTQTPSPVSAAVGDTVTIKCQSSQSVYKNNYLSWYQQKPGQPPKLLIYDASNLPSG 102 2016273912 14 Dec 2016 VPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGDYDDDTDNGFGGGTEVVVKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 142).

[00432] The invention further includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QS VEESGGRLVTPGTPLTLTCT VSGIDLS S Y AMI WVRQ APGKGLEYIGIIWSGGTYY ATW AKGRFTISKTSTTVDLQITSPTTEDAATYFCAAGGGSIYDVWGPGTLVTVSS (SEQ ID NO: 143).

[00433] The invention also includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: Q S VEES GGRL VTPGTPLTLT CT V S GIDLS S Y AMI WVRQ APGKGLEYIGII W S GGT Y Y ATW AKGRFTISKTSTTVDLQITSPTTEDAATYFCAAGGGSIYDVWGPGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRV V S VLTVLHQD WLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 144).

[00434] The invention further contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 145; SEQ ID NO: 146; and SEQ ID NO: 147 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 141 or the light chain sequence of SEQ ID NO: 142, and/or one or more of the polypeptide sequences of SEQ ID NO: 148; SEQ ID NO: 149; and SEQ ID NO: 150 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 143 or the heavy chain sequence of SEQ ID NO: 144, or combinations of these polypeptide sequences. In another embodiment of the invention, 103 2016273912 14 Dec 2016 the antibodies of the invention or fragments thereof for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00435] The invention also contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 141 or SEQ ID NO: 142. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 143 or SEQ ID NO: 144.

[00436] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 145; SEQ ID NO: 146; and SEQ ID NO: 147 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 141 or the light chain sequence of SEQ ID NO: 142.

[00437] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 148; SEQ ID NO: 149; and SEQ ID NO: 150 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 143 or the heavy chain sequence of SEQ ID NO: 144.

[00438] The invention also contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 141; the variable heavy chain region of SEQ ID NO: 143; the complementarity-determining regions (SEQ ID NO: 145; SEQ 104 2016273912 14 Dec 2016 ID NO: 146; and SEQ ID NO: 147) of the variable light chain region of SEQ ID NO: 141; and the complementarity-determining regions (SEQ ID NO: 148; SEQ ID NO: 149; and SEQ ID NO: 150) of the variable heavy chain region of SEQ ID NO: 143.

[00439] In a particularly preferred embodiment of the invention, the chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl5, comprising, or alternatively consisting of, SEQ ID NO: 142 and SEQ ID NO: 144, and having at least one of the biological activities set forth herein.

[00440] In a further particularly preferred embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl5, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 141 and the variable heavy chain sequence of SEQ ID NO: 143 or comprises another Fab that binds to the same or overlapping epitope as Ab 15,. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 141 and/or SEQ ID NO: 143 in said Fab while retaining binding specificity for NGF.

[00441] In one embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl5. In another embodiment of the invention, anti-NGF antibodies for treatment or prevention of pain and pain associated conditions such as Abl5 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00442] Antibody Ab 16 [00443] The invention contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NS AID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is Abl6 or fragments thereof, for example as set forth below, or comprises 105 2016273912 14 Dec 2016 another antibody or antibody fragment that binds to the same or overlapping epitope as Abl6, in a therapeutically effective amount which inhibits the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: ALVMTQTPSSTSEPVGGTVTINCQASQNIGNDLSWYQQKPGQPPELLIYSTSKLATGVPK RFSGSRSGTQFTLTISDLECDDAATYYCLGVYSYISDDGNAFGGGTEVVVKR (SEQ ID NO: 151).

[00444] The invention also includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: ALVMTQTPSSTSEPVGGTVTINCQASQNIGNDLSWYQQKPGQPPELLIYSTSKLATGVPK RFSGSRSGTQFTLTISDLECDDAATYYCLGVYSYISDDGNAFGGGTEVWKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 152).

[00445] The invention further includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QSVEEFGGRLVTPGTPLTLTCTVSGFSLNNYAMTWVRQAPGKGLEWIGIIGSIGTTYYAS WAKGRFFISKTSTTVDLKIISPTTEDTATYFCARDAGVTVDGYGYYFNIWGPGTLVTVSS (SEQ ID NO: 153).

[00446] The invention also includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QSVEEFGGRLVTPGTPLTLTCTVSGFSLNNYAMTWVRQAPGKGLEWIGIIGSIGTTYYAS WAKGRFFISKTSTTVDLKIISPTTEDTATYFCARDAGVTVDGYGYYFNIWGPGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL Y SLS S VVTVPS S SLGT QT YICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCP APELLGG 106 2016273912 14 Dec 2016 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY ASTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 154).

[00447] The invention further contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 155; SEQ ID NO: 156; and SEQ ID NO: 157 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 151 or the light chain sequence of SEQ ID NO: 152, and/or one or more of the polypeptide sequences of SEQ ID NO: 158; SEQ ID NO: 159; and SEQ ID NO: 160 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 153 or the heavy chain sequence of SEQ ID NO: 154, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00448] The invention also contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 151 or SEQ ID NO: 152. In another embodiment of the invention, antibody fragments of the invention for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 153 or SEQ ID NO: 154.

[00449] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 155; SEQ ID NO: 156; and SEQ ID NO: 157 which correspond to the complementarity-107 2016273912 14 Dec 2016 determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 151 or the light chain sequence of SEQ ID NO: 152.

[00450] In a further embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 158; SEQ ID NO: 159; and SEQ ID NO: 160 which correspond to the complementaritydetermining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 153 or the heavy chain sequence of SEQ ID NO: 154.

[00451] The invention also contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 151; the variable heavy chain region of SEQ ID NO: 153; the complementarity-determining regions (SEQ ID NO: 155; SEQ ID NO: 156; and SEQ ID NO: 157) of the variable light chain region of SEQ ID NO: 151; and the complementarity-determining regions (SEQ ID NO: 158; SEQ ID NO: 159; and SEQ ID NO: 160) of the variable heavy chain region of SEQ ID NO: 153.

[00452] In a particularly preferred embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl6, comprising, or alternatively consisting of, SEQ ID NO: 152 and SEQ ID NO: 154, and having at least one of the biological activities set forth herein.

[00453] In a further particularly preferred embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl6, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 151 and the variable heavy chain sequence of SEQ ID NO: 15 3 or comprises another Fab or another bivalent or monovalent antibody fragment that binds to the same or overlapping epitope as Abl6. This embodiment of the invention further contemplates 108 2016273912 14 Dec 2016 additions, deletions, and variants of SEQ ID NO: 151 and/or SEQ ID NO: 153 in said Fab while retaining binding specificity for NGF.

[00454] In one embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl6. In another embodiment of the invention, anti-NGF antibodies for treatment or prevention of pain and pain associated conditions such as Abl6 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00455] Antibody Ab 17 [00456] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is Abl7 or fragments thereof, for example as set forth below, or comprises another antibody or antibody fragment that binds to the same or overlapping epitope as Abl7, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AIEMTQTPFSVSAAVGGTVTIKCQASQTISNYLAWYQQKPGQPPKLLIYGASNLESGVPS RFKGSGSGTQFTLTISDLECDDAATYYCQQGYTISNVDNNVFGGGTEVWKR (SEQ ID NO: 161).

[00457] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AIEMTQTPFSVSAAVGGTVTIKCQASQTISNYLAWYQQKPGQPPKLLIYGASNLESGVPS RFKGSGSGTQFTLTISDLECDDAATYYCQQGYTISNVDNNVFGGGTEVWKRTVAAPSV 109 2016273912 14 Dec 2016 FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 162).

[00458] The invention further optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGGSLTLTCAASGFSLTGYNLVWVRQAPGKGLEWIGFISYGDTTYYA SWAKGRFTISKTSTTVTLTITDLQPSDTGTYFCARETANTYDYGIWGPGTLVTVSS (SEQ ID NO: 163).

[00459] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGGSLTLTCAASGFSLTGYNLVWVRQAPGKGLEWIGFISYGDTTYYA SWAKGRFTISKTSTTVTLTITDLQPSDTGTYFCARETANTYDYGIWGPGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSYYTYPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRVV SVLTVLHQD WLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKN Q VSLT CL VKGF YP SDIAVE WE SN GQPENN YKTTPP VLDSDGSFFL Y SKLT VDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 164).

[00460] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 165; SEQ ID NO: 166; and SEQ ID NO: 167 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 161 or the light chain sequence of SEQ ID NO: 162, and/or one or more of the polypeptide sequences of SEQ ID NO: 168; SEQ ID NO: 169; and SEQ ID NO: 170 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 163 or the heavy chain sequence of SEQ ID NO: 164, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof for treatment 110 2016273912 14 Dec 2016 or prevention of pain and pain associated conditions comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00461] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 161 or SEQ ID NO: 162. In another optional embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 163 or SEQ ID NO: 164.

[00462] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 165; SEQ ID NO: 166; and SEQ ID NO: 167 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 161 or the light chain sequence of SEQ ID NO: 162.

[00463] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 168; SEQ ID NO: 169; and SEQ ID NO: 170 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 163 or the heavy chain sequence of SEQ ID NO: 164.

[00464] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 161; the variable heavy chain region of SEQ ID NO: 163; the complementarity-determining regions (SEQ ID NO: 165; SEQ ID NO: 166; and SEQ ID NO: 167) of the variable light chain region of SEQ ID NO: 161; and 111 2016273912 14 Dec 2016 the complementarity-determining regions (SEQ ID NO: 168; SEQ ID NO: 169; and SEQ ID NO: 170) of the variable heavy chain region of SEQ ID NO: 163.

[00465] In a particularly preferred optional embodiment of the invention, the chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl7, comprising, or alternatively consisting of, SEQ ID NO: 162 and SEQ ID NO: 164, and having at least one of the biological activities set forth herein.

[00466] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl7, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 161 and the variable heavy chain sequence of SEQ ID NO: 163 or comprises another Fab or monovalent or bivalent antibody fragment that binds to the same or overlapping epitope as Ab 15,. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 161 and/or SEQ ID NO: 163 in said Fab while retaining binding specificity for NGF.

[00467] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl7. In another embodiment of the invention, anti-NGF antibodies such as Abl7 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00468] Antibody Ab 18 [00469] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is Abl8 or fragments thereof, for example as set forth below, or comprises another antibody or antibody fragment that binds to the same or overlapping epitope 112 2016273912 14 Dec 2016 as Abl8, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQASQTISNYLAWYQQKPGKAPKLLIYGASNLESGVPS RFSGSGSGTEFTLTIS SLQPDDFATYYCQQGYTISNVDNNVFGGGTKVEIKR (SEQ ID NO: 171).

[00470] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQASQTISNYLAWYQQKPGKAPKLLIYGASNLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQGYTISNVDNNVFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 172).

[00471] The invention further optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSGYNLVWVRQAPGKGLEWVGFISYGDTTY Y ASS AKGRFTISRDNSKNTLYLQMNSLRAEDT AVYY CARET ANTYD Y GIWGQGTLVTV SS (SEQ ID NO: 173).

[00472] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLV QPGGSLRLSC AASGFTV SGYNLV WVRQ APGKGLEWV GFIS Y GDTTY YASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETANTYDYGIWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE 113 2016273912 14 Dec 2016 QY ASTYRW S VLTVLHQD WLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 174).

[00473] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 175; SEQ ID NO: 176; and SEQ ID NO: 177 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 171 or the light chain sequence of SEQ ID NO: 172, and/or one or more of the polypeptide sequences of SEQ ID NO: 178; SEQ ID NO: 179; and SEQ ID NO: 180 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 173 or the heavy chain sequence of SEQ ID NO: 174, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00474] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 171 or SEQ ID NO: 172. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 173 or SEQ ID NO: 174.

[00475] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 175; SEQ ID NO: 176; and SEQ ID NO: 177 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 171 or the light chain sequence of SEQ ID NO: 172. 114 2016273912 14 Dec 2016 [00476] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 178; SEQ ID NO: 179; and SEQ ID NO: 180 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 173 or the heavy chain sequence of SEQ ID NO: 174.

[00477] The invention also optional contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 171; the variable heavy chain region of SEQ ID NO: 173; the complementarity-determining regions (SEQ ID NO: 175; SEQ ID NO: 176; and SEQ ID NO: 177) of the variable light chain region of SEQ ID NO: 171; and the complementarity-determining regions (SEQ ID NO: 178; SEQ ID NO: 179; and SEQ ID NO: 180) of the variable heavy chain region of SEQ ID NO: 173.

[00478] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl8, comprising, or alternatively consisting of, SEQ ID NO: 172 and SEQ ID NO: 174, and having at least one of the biological activities set forth herein.

[00479] In a further particularly preferred optional embodiment of the invention, antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl8, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 171 and the variable heavy chain sequence of SEQ ID NO: 173 or comprise another Fab or antibody fragment that binds to the same or overlapping epitope as Abl8. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 171 and/or SEQ ID NO: 173 in said Fab while retaining binding specificity for NGF. 115 2016273912 14 Dec 2016 [00480] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl8. In another embodiment of the invention, anti-NGF antibodies such as Abl8 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00481] Antibody Abl 9 [00482] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is Abl9 or fragments thereof, for example as set forth below, or comprises another antibody or antibody fragment that binds to the same or overlapping epitope as Abl 9, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: AAVLTQTPSPVSAAVGGTVSISCQSSQNVYKNNYLSWYQQKPGQPPKLLIYKASTLASG VPSRFKGSGSGTDFTLTISDVQCDAAATYYCAGGYSSSSDNAFGGGTEVVVKR (SEQ ID NO: 181).

[00483] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: AAVLTQTPSPVSAAVGGTVSISCQSSQNVYKNNYLSWYQQKPGQPPKLLIYKASTLASG VPSRFKGSGSGTDFTLTISDVQCDAAATYYCAGGYSSSSDNAFGGGTEVVVKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 182). 116 2016273912 14 Dec 2016 [00484] The invention further optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: QSVEASGGRLVMPGGSLTLTCTASGFSLSTYWMSWVRQAPGKGLEWIGDIYFSNEETN YATWAKGRFTISKTSTTVDLNVISPTTEDTATYFCARGSPDVEIAIDMWGQGTLVTVSS (SEQ ID NO: 183).

[00485] The invention also optionally includes chimeric antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: QSVEASGGRLVMPGGSLTLTCTASGFSLSTYWMSWVRQAPGKGLEWIGDIYFSNEETN YATWAKGRFTISKTSTTVDLNVISPTTEDTATYFCARGSPDVEIAIDMWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG L Y SLS S V VTVPS S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 184).

[00486] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 185; SEQ ID NO: 186; and SEQ ID NO: 187 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 181 or the light chain sequence of SEQ ID NO: 182, and/or one or more of the polypeptide sequences of SEQ ID NO: 188; SEQ ID NO: 189; and SEQ ID NO: 190 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 183 or the heavy chain sequence of SEQ ID NO: 184, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them. 117 2016273912 14 Dec 2016 [00487] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 181 or SEQ ID NO: 182. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 183 or SEQ ID NO: 184.

[00488] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 185; SEQ ID NO: 186; and SEQ ID NO: 187 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 181 or the light chain sequence of SEQ ID NO: 182.

[00489] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 188; SEQ ID NO: 189; and SEQ ID NO: 190 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 183 or the heavy chain sequence of SEQ ID NO: 184.

[00490] The invention also optionally contemplates antibody fragments which include one or more of the antibody fragments described herein for treatment or prevention of pain and pain associated conditions. In one optional embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 181; the variable heavy chain region of SEQ ID NO: 183; the complementarity-determining regions (SEQ ID NO: 185; SEQ ID NO: 186; and SEQ ID NO: 187) of the variable light chain region of SEQ ID NO: 181; and the complementarity-determining regions (SEQ ID NO: 188; SEQ ID NO: 189; and SEQ ID NO: 190) of the variable heavy chain region of SEQ ID NO: 183.

[00491] In a particularly preferred optional embodiment of the invention, the chimeric anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Abl9, 118 2016273912 14 Dec 2016 comprising, or alternatively consisting of, SEQ ID NO: 182 and SEQ ID NO: 184, and having at least one of the biological activities set forth herein.

[00492] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl9, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 181 and the variable heavy chain sequence of SEQ ID NO: 183 or comprises another Fab or antibody fragment that binds to the same or overlapping epitope as Abl9. This optional embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 181 and/or SEQ ID NO: 183 in said Fab while retaining binding specificity for NGF.

[00493] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl9. In another embodiment of the invention, anti-NGF antibodies such as Abl9 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00494] Antibody Ab20 [00495] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is Ab20 or fragments thereof, for example as set forth below, or comprises another antibody or antibody fragment that binds to the same or overlapping epitope as Ab20, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: 119 2016273912 14 Dec 2016 DIQMTQSPSSLSASVGDRVTITCQSSQNVYKNNYLSWYQQKPGKVPKLLIYKASTLASG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYTSSSDNAFGGGTKVEIKR (SEQ ID NO: 191).

[00496] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: DIQMTQSPSSLSASVGDRVTITCQSSQNVYKNNYLSWYQQKPGKVPKLLIYKASTLASG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYTSSSDNAFGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 192).

[00497] The invention further optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSTYWMSWVRQAPGKGLEWVGDIYFSNEET NYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSPDYEIAIDMWGQGTLV TVSS (SEQ ID NO: 193).

[00498] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSTYWMSWVRQAPGKGLEWVGDIYFSNEET NY AT S AKGRFTISRDN SKNTLYLQMN SLRAEDT A V Y Y C ARGSPD VEIAIDM WGQGTL V TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY ASTYRVV S VLTVLHQDWLNGKEYKCKV SNKALP APIEKTISKAKGQPREPQVYTL PPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 194).

[00499] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide 120 2016273912 14 Dec 2016 sequences of SEQ ID NO: 195; SEQ ID NO: 196; and SEQ ID NO: 197 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 191 or the light chain sequence of SEQ ID NO: 192, and/or one or more of the polypeptide sequences of SEQ ID NO: 198; SEQ ID NO: 199; and SEQ ID NO: 200 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 193 or the heavy chain sequence of SEQ ID NO: 194, or combinations of these polypeptide sequences. In another embodiment of the invention, the antibodies of the invention or fragments thereof for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00500] The invention also optionally contemplates fragments of the antibody having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 191 or SEQ ID NO: 192. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 193 or SEQ ID NO: 194.

[00501] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 195; SEQ ID NO: 196; and SEQ ID NO: 197 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 191 or the light chain sequence of SEQ ID NO: 192.

[00502] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 198; SEQ ID NO: 199; and SEQ ID NO: 200 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 193 or the heavy chain sequence of SEQ ID NO: 194. 121 2016273912 14 Dec 2016 [00503] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 191; the variable heavy chain region of SEQ ID NO: 193; the complementarity-determining regions (SEQ ID NO: 195; SEQ ID NO: 196; and SEQ ID NO: 197) of the variable light chain region of SEQ ID NO: 191; and the complementarity-determining regions (SEQ ID NO: 198; SEQ ID NO: 199; and SEQ ID NO: 200) of the variable heavy chain region of SEQ ID NO: 193.

[00504] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab20, comprising, or alternatively consisting of, SEQ ID NO: 192 and SEQ ID NO: 194, and having at least one of the biological activities set forth herein.

[00505] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab20, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 191 and the variable heavy chain sequence of SEQ ID NO: 193. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 191 and/or SEQ ID NO: 193 in said Fab while retaining binding specificity for NGF.

[00506] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab20. In another embodiment of the invention, anti-NGF antibodies for treatment or prevention of pain and pain associated conditions such as Ab20 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00507] Antibody Ab21 122 2016273912 14 Dec 2016 [00508] The invention optionally contemplates methods of treating pain and the specific pain associated disorders alone or is association with another active agent, e.g., an NSAID or opioid analgesic, wherein the antibodies include chimeric antibodies having binding specificity to NGF wherein the antibody is Ab21 or fragments thereof, or another antibody or antibody fragment that binds to the same or overlapping epitope as Ab5, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75. In one embodiment, the invention includes chimeric or humanized antibodies having binding specificity to NGF and possessing a variable light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKR (SEQ ID NO: 51).

[00509] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 401).

[00510] The invention further optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT WY AS S ARGRFTISRDNSKNTLYLQMNSLRAEDT AV YY CARGY GRSV AYYVFNIWGPGT LVTVSS (SEQ ID NO: 53).

[00511] The invention also optionally includes chimeric or humanized antibodies for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below:

E VQL VE S GGGL V QPGGSLRLS C AAS GFT V SN Y AV G WVRQ APGKGLE WV GIIGRN GNT 123 2016273912 14 Dec 2016

WYAS S ARGRFTISRDNSKNTLYLQMNSLRAEDT AVYY C ARGY GRS V AYYVFNIWGPGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPA

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 402).

[00512] The invention further optionally contemplates antibodies for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 401, and/or one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 402, or combinations of these polypeptide sequences. In another optional embodiment of the invention, the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00513] The invention also optionally contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ ID NO: 401. In another embodiment of the invention, antibody fragments of the invention for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 53 or SEQ ID NO: 402.

[00514] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated 124 2016273912 14 Dec 2016 conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 401.

[00515] In a further optional embodiment of the invention, fragments of the antibody having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 402.

[00516] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 51; the variable heavy chain region of SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60) of the variable heavy chain region of SEQ ID NO: 53.

[00517] In a particularly preferred optional embodiment of the invention, the chimeric or humanized anti-NGF antibody for treatment or prevention of pain and pain associated conditions is Ab21, comprising, or alternatively consisting of, SEQ ID NO: 401 and SEQ ID NO: 402, and having at least one of the biological activities set forth herein.

[00518] In a further particularly preferred optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab21, the Fab fragment includes the variable light chain sequence of SEQ ID NO: 51 and the variable heavy chain sequence of SEQ ID NO: 53 or another Fab or monovalent antibody fragment that binds to the same or overlapping epitope as 125 2016273912 14 Dec 2016

Ab5,. This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 51 and/or SEQ ID NO: 53 in said Fab while retaining binding specificity for NGF.

[00519] In one optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Ab21. In another embodiment of the invention, anti-NGF antibodies for treatment or prevention of pain and pain associated conditions such as Ab21 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00520] Antibody fragment Fabl [00521] The invention optionally contemplates methods of treating pain using antibody fragment Fabl or fragments thereof, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75.

In one embodiment, the invention optionally includes Fab antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below: [00522] DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 405).

[00523] The invention further optionally includes Fab antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below:

E VQL VE S GGGL V QPGGSLRLS C AAS GFT V SN Y AV G WVRQ APGKGLE WV GIIGRN GNT WYAS S ARGRFTISRDNSKNTLYLQMNSLRAEDT AVYY C ARGY GRS V AYYVFNIWGPGT 126 2016273912 14 Dec 2016 LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQS SGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKVDARVEPKSCDKTH (SEQ ID NO: 406).

[00524] The invention further optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 405, and/or one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 406, or combinations of these polypeptide sequences. In another optional embodiment of the invention, antibody fragments of the invention for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00525] The invention also optionally contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ ID NO: 405. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 53 or SEQ ID NO: 406.

[00526] In a further optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 405. 127 2016273912 14 Dec 2016 [00527] In a further optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 406.

[00528] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 51; the variable heavy chain region of SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60) of the variable heavy chain region of SEQ ID NO: 53.

[00529] In a particularly preferred optional embodiment of the invention, the anti-NGF antibody fragment for treatment or prevention of pain and pain associated conditions is Fabl, comprising SEQ ID NO: 405 and SEQ ID NO: 406, or another Fab or antibody fragment that binds to the same or overlapping epitope as Fabl, and having at least one of the biological activities set forth herein. In one embodiment of the invention, antibody fragment Fabl may be produced by enzymatic digestion (e.g., papain) of Ab21.

[00530] Antibody fragment Fab2 [00531] The invention optionally contemplates methods of treating pain using antibody fragment Fab2 or fragments thereof, for example as set forth below, in a therapeutically effective amount which inhibits the association of NGF with TrkA and the association of NGF with p75.

In one embodiment, the invention includes Fab antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a light chain sequence comprising the sequence set forth below:

DIQMTQSPSTLSASVGDRVTITCQASQSIYSNLAWYQQKPGKAPKLLIYDASTLESGVPS 128 2016273912 14 Dec 2016 RFSGSGSGTEYTLTISSLQPDDFATYYCQQGFTVSDIDNAFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 407).

[00532] The invention further optionally includes Fab antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF and possessing a heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYAVGWVRQAPGKGLEWVGIIGRNGNT WYASSARGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGRSVAYYVFNIWGPGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTH (SEQ ID NO: 408).

[00533] The invention further optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions comprising one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 407, and/or one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 408, or combinations of these polypeptide sequences. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.

[00534] The invention also optionally contemplates fragments of the antibody for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, antibody fragments of the invention for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ ID NO: 407. In another embodiment of the invention, antibody fragments of the invention for treatment or prevention of pain and pain 129 2016273912 14 Dec 2016 associated conditions comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 53 or SEQ ID NO: 408.

[00535] In a further optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 407.

[00536] In a further optional embodiment of the invention, antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 408.

[00537] The invention also optionally contemplates antibody fragments for treatment or prevention of pain and pain associated conditions which include one or more of the antibody fragments described herein. In one embodiment of the invention, fragments of the antibodies having binding specificity to NGF for treatment or prevention of pain and pain associated conditions comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 51; the variable heavy chain region of SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51; and the complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60) of the variable heavy chain region of SEQ ID NO: 53.

[00538] In a particularly preferred optional embodiment of the invention, the anti-NGF antibody fragment for treatment or prevention of pain and pain associated conditions is Fab2, comprising SEQ ID NO: 407 and SEQ ID NO: 408, or another Fab or antibody fragment that binds to the same or overlapping epitope as Fab2, and having at least one of the biological activities set forth herein. 130 2016273912 14 Dec 2016 [00539] In another optional embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention, antibody fragment Fab2 may be produced by expression in Pichia pastoris using protocols set forth herein in the examples.

[00540] In another embodiment, antibody fragments may be present in one or more of the following non-limiting forms: Fab, Fab', F(ab’)2, Fv and single chain Fv antibody forms. In a preferred embodiment, the anti-NGF antibodies described herein further comprises the kappa constant light chain sequence comprising the sequence set forth below: [00541] V AAP S VFIFPP SDEQLKS GT AS V VCLLNNF YPRE AKV Q WKVDN ALQ S GN S QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 412).

[00542] In another preferred optional embodiment, the anti-NGF antibodies described herein for treatment or prevention of pain and pain associated conditions further comprises the gamma-1 constant heavy chain polypeptide sequence comprising the sequence set forth below: [00543] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVV SVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 413).

[00544] In another optional embodiment, the invention contemplates an isolated anti-NGF antibody for treatment or prevention of pain and pain associated conditions comprising a Vh polypeptide sequence selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, or 402, or a variant thereof; and further comprising a VL polypeptide sequence selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 131 2016273912 14 Dec 2016 121, 131, 141, 151, 161, 171, 181, 191, or 401, or a variant thereof, wherein one or more of the framework residues (FR residues) in said Vh or VL polypeptide has been substituted with another amino acid residue resulting in an anti-NGF antibody that specifically binds NGF. The invention contemplates humanized and chimeric forms of these antibodies for treatment or prevention of pain and pain associated conditions. The chimeric antibodies may include an Fc derived from IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgGlO, IgGl 1, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19 constant regions.

[00545] In one embodiment of the invention, the antibodies or Vh or VL polypeptides originate or are selected from one or more rabbit B cell populations prior to initiation of the humanization process referenced herein.

[00546] In another embodiment of the invention, the anti-NGF antibodies and fragments thereof for treatment or prevention of pain and pain associated conditions do not have binding specificity for p75 or TrkA. In a further embodiment of the invention, there is contemplated methods for treating pain comprising using the anti-NGF antibodies and fragments thereof to inhibit the association of NGF with p75 and/or TrkA. In another embodiment of the invention, there is contemplated methods for treating pain comprising using anti-NGF antibodies and fragments thereof to inhibit the association of NGF with TrkA and/or multimers thereof and/or antagonizes the biological effects thereof. In another embodiment of the invention, there is contemplated methods for treating pain comprising using anti-NGF antibodies and fragments thereof to inhibit the association of NGF with p75 and/or multimers thereof and the association of NGF with TrkA and/or multimers thereof, and antagonizes the biological effects of p75 and TrkA.

[00547] As stated supra, antibodies and fragments thereof may be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.

[00548] Regarding detectable moieties, further exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase 132 2016273912 14 Dec 2016 and luciferase. Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dansyl chloride. Further exemplary chemiluminescent moieties include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Further exemplary radioactive materials include, but are not limited to, Iodine 125 (125I), Carbon 14 (14C), Sulfur 35 (35S), Tritium (3H) and Phosphorus 32 (32P).

[00549] Regarding functional moieties, exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimytotic agents such as the vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (0,P'-(DDD)), interferons, and mixtures of these cytotoxic agents.

[00550] Further cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin. Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be conjugated to the humanized or chimeric antibodies, or binding fragments thereof, to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419(1980)). 133 2016273912 14 Dec 2016 [00551] Other cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled to the antibody, or binding fragments thereof, with or without the use of a complex-forming agent. Such radionuclides include beta-emitters such asPhosphorus-32 (32P), Scandium-47 (47Sc), Copper-67 (67Cu), Gallium-67 (67Ga), Yttrium-88 (88Y), Yttrium-90 (90Y), Iodine-125 (125I), Iodine-131 (131I), Samarium-153 (153Sm), Lutetium-177 (177Lu), Rhenium-186 (186Re) or Rhenium-188 (188Re), and alpha-emitters such as Astatine-211 (211At), Lead-212 (212Pb), Bismuth-212 (212Bi) or -213 (213Bi) or Actinium-225 (225Ac).

[00552] Further exemplary radioactive materials include, but are not limited to, Iodine 125 (125I), Carbon 14 (14C), Sulfur 35 (35S), Tritium (3H) and Phosphorus 32 (32P).

[00553] Regarding functional moieties, exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimytotic agents such as the vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (0,P'-(DDD)), interferons, and mixtures of these cytotoxic agents.

[00554] Further cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin. Toxic 134 2016273912 14 Dec 2016 enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be conjugated to the humanized or chimeric antibodies, or binding fragments thereof, to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).

[00555] Other cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled to the antibody, or binding fragments thereof, with or without the use of a complex-forming agent. Such radionuclides include beta-emitters such asPhosphorus-32 (32P), Scandium-47 (47Sc), Copper-67 (67Cu), Gallium-67 (67Ga), Yttrium-88 (88Y), Yttrium-90 (90Y), Iodine-125 (125I), Iodine-131 (131I), Samarium-153 (153Sm), Lutetium-177 (177Lu), Rhenium-186 (186Re) or Rhenium-188 (188Re), and alpha-emitters such as Astatine-211 (211At), Lead-212 (212Pb), Bismuth-212 (212Bi) or -213 (213Bi) or Actinium-225 (225Ac)..

[00556] Methods are known in the art for conjugating an antibody or binding fragment thereof to a detectable moiety and the like, such as for example those methods described by Hunter et al, Nature 144:945 (1962); David et al, Biochemistry 13:1014 (1974); Pain et al, J. Immunol. Meth. 40:219 (1981); andNygren, J., Histochem. and Cytochem. 30:407 (1982).

[00557] Embodiments described herein further include variants and equivalents that are substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variable regions and CDRs set forth herein. These may contain, e.g., conservative substitution mutations, (i.e., the substitution of one or more amino acids by similar amino acids). For example, conservative substitution refers to the substitution of an amino acid with another within the same general class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art.

[00558] In another embodiment, the invention contemplates polypeptide sequences having at least 90% or greater sequence homology to any one or more of the polypeptide sequences of 135 2016273912 14 Dec 2016 antibody fragments, variable regions and CDRs set forth herein. More preferably, the invention contemplates polypeptide sequences having at least 95% or greater sequence homology, even more preferably at least 98% or greater sequence homology, and still more preferably at least 99% or greater sequence homology to any one or more of the polypeptide sequences of antibody fragments, variable regions and CDRs set forth herein. Methods for determining homology between nucleic acid and amino acid sequences are well known to those of ordinary skill in the art.

[00559] In another embodiment, the invention further contemplates the above-recited polypeptide homologs of the antibody fragments, variable regions and CDRs set forth herein further having anti-NGF activity. Non-limiting examples of anti-NGF activity are set forth herein.

[00560] In another embodiment, the invention further contemplates the generation and use of anti-idiotypic antibodies that bind any of the foregoing sequences. In an exemplary embodiment, such an anti-idiotypic antibody could be administered to a subject who has received an anti-NGF antibody to modulate, reduce, or neutralize, the effect of the anti-NGF antibody. Such anti-idiotypic antibodies could also be useful for treatment of an autoimmune disease characterized by the presence of anti-NGF antibodies. A further exemplary use of such anti-idiotypic antibodies is for detection of the anti-NGF antibodies of the present invention, for example to monitor the levels of the anti-NGF antibodies present in a subject’s blood or other bodily fluids.

[00561] The present invention also contemplates anti-NGF antibodies comprising any of the polypeptide or polynucleotide sequences described herein substituted for any of the other polynucleotide sequences described herein. For example, without limitation thereto, the present invention contemplates antibodies comprising the combination of any of the variable light chain and variable heavy chain sequences described herein, and further contemplates antibodies resulting from substitution of any of the CDR sequences described herein for any of the other CDR sequences described herein.

[00562] 136 2016273912 14 Dec 2016 [00563] Polynucleotides Encoding Anti-NGF Antibody Polypeptides [00564] Antibody Abl [00565] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Abl polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl polypeptides. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 1: [00566] GCCCTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGA GGC AC AGT C ACC ATC AATTGCC AGGCC AGT C AGAAC ATTT AC AGC AATTT AGCCT GG T AT C AAC AG AG AC C AGGGC AGCGT CCC AAGCT CCT G ATCT AT GGT GC AT CC AAT CT G GAT GCT GGGGTCCC AT C GCGGTT C AGAGGC AGTGG AT CT GGG AC AG AGT AC ACT CT CACCATCAGCGACCTGGAGTGTGACGATGTTGGCACTTACTACTGTCAAAGTGCTTT T GAT AGT GAT AGT ACT G A AAAT ACTTTC GGC GG AGGG ACC G AGGT GGT GGTC AAAC GT (SEQ ID NO: 201).

[00567] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 2:

[00568] GCCCTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGA GGC AC AGT C ACC ATC AATT GC C AGGCC AGT C AG AAC ATTT AC AGC AATTT AGC CTGG TATCAACAGAGACCAGGGCAGCGTCCCAAGCTCCTGATCTATGGTGCATCCAATCTG GAT GCT GGGGTCCC ATCGCGGTTC AGAGGC AGTGGAT CT GGGAC AGAGT AC ACT CT CACCATCAGCGACCTGGAGTGTGACGATGTTGGCACTTACTACTGTCAAAGTGCTTT T GAT AGT GAT AGT ACT G A AAAT ACTTTC GGC GG AGGG ACC G AGGT GGT GGT C AAAC GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG 137 2016273912 14 Dec 2016 T AC AGT GGAAGGT GGAT AACGCCCTCC AATCGGGT AACTCCC AGGAGAGTGTC AC A GAGC AGGAC AGC AAGGAC AGC ACCT AC AGCCT C AGC AGC ACCCT GACGCT GAGC AA AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 202).

[00569] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 3: [00570] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGTCTCTGGCTTCTCCCTCAGTAGCTATGCAATGAGCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTACTAGTATTG GTAGCACAGTCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG ACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT CT GT GCC AG AGGCT AC GAT G ACT AT GAT GAG AT G ACCT ACTTT A AC AT CT GGGGCC A GGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 203).

[00571] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 4:

[00572] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGTCTCTGGCTTCTCCCTCAGTAGCTATGCAATGAGCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTACTAGTATTG GTAGCACAGTCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG ACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT CT GT GCC AGAGGCT ACGAT GACT AT GATGAGATGACCTACTTT A AC AT CT GGGGCC A GGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCT GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT C AC AAGC CC AGC AAC AC C AAGGT GGAC AAGAG AGTT G AGCCC AAAT CTT GT GAC AA 138 2016273912 14 Dec 2016

AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT

CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC

ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG

TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGC

CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG

CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA

CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA

TCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT

CT AT CCC AGCG AC ATCGCCGT GGAGT GGGAG AGC AAT GGGC AGCCGGAGAAC AACT

ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC

TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG

CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA TGA (SEQ ID NO: 204).

[00573] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 205; SEQ ID NO: 206; and SEQ ID NO: 207 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2.

[00574] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 208; SEQ ID NO: 209; and SEQ ID NO: 210 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4.

[00575] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or 139 2016273912 14 Dec 2016 prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 201 encoding the light chain variable sequence of SEQ ID NO: 1; the polynucleotide SEQ ID NO: 202 encoding the light chain sequence of SEQ ID NO: 2; the polynucleotide SEQ ID NO: 203 encoding the heavy chain variable sequence of SEQ ID NO: 3; the polynucleotide SEQ ID NO: 204 encoding the heavy chain sequence of SEQ ID NO: 4; polynucleotides encoding the complementaritydetermining regions (SEQ ID NO: 205; SEQ ID NO: 206; and SEQ ID NO: 207) of the light chain variable sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 208; SEQ ID NO: 209; and SEQ ID NO: 210) of the heavy chain variable sequence of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4.

[00576] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments for treatment or prevention of pain and pain associated conditions having binding specificity for NGF. With respect to antibody Abl, the polynucleotides encoding the full length Abl antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 202 encoding the light chain sequence of SEQ ID NO: 2 and the polynucleotide SEQ ID NO: 204 encoding the heavy chain sequence of SEQ ID NO: 4.

[00577] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments for treatment or prevention of pain and pain associated conditions may be produced by enzymatic digestion (e.g., papain) of Abl following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl or Fab fragments thereof may be produced via expression of Abl polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example 140 2016273912 14 Dec 2016 diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00578] Antibody Ab2 [00579] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Ab2 polypeptides which inhibit the association of NGF with TrkA and the association of NGF with p75, for treatment or prevention of pain and pain associated conditions having binding specificity to NGF in methods of treating pain in an individual comprising administering to said individual antibody Ab2 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides having binding specificity to NGF for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 11: [00580] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GAC AGAGTC ACC AT C ACTT GCC AGGCC AGTC AGAAC ATTT AC AGC AACTT AGCCT G GTATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATCT GG AT GCT GG AGTCC C AT C AAGGTT CTCT GGC AGT GG ATCTGGG AC AG AGT AC ACT CT CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAAAGTGCTTT T GAT AGT GAT AGT ACT G AAAAC ACTTTCGGC GG AGG AACC AAGGT GG AAAT C AAAC GT (SEQ ID NO: 211).

[00581] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 12:

[00582] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAACATTTACAGCAACTTAGCCTG GTATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATCT GGAT GCT GGAGTCCCATC AAGGTT CT CT GGC AGTGGATCTGGGAC AGAGT AC ACTCT CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAAAGTGCTTT 141 2016273912 14 Dec 2016

T GAT AGT GAT AGT ACT G AAAAC ACTTTCGGC GG AGG AACC AAGGT GG AAAT C AAAC

GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT

CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG

T AC AGT GGAAGGT GGAT AACGCCCTCC AATCGGGT AACTCCC AGGAGAGTGT C AC A

GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA

AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 212).

[00583] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 13: [00584] G AGGT GC AGCTGGTGG AGT CT GGGGG AGGCTT GGT CC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCTATGCAATGAG CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTACTAGTA TTGGTAGCACAGTCTACGCGAGCAGCGCGAAAGGCCGATTCACCATCTCCAGAGAC AATT C C AAG AAC AC CCT GT AT CTTC AAAT GAAC AGCCT G AG AGCT G AGG AC ACT GCT GTGTATTACTGTGCTAGAGGCTACGATGACTATGATGAGATGACCTACTTTAACATC TGGGGCCAAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 213).

[00585] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 14:

[00586] GAGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGTCC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCTATGCAATGAG CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTACTAGTA TTGGTAGCACAGTCTACGCGAGCAGCGCGAAAGGCCGATTCACCATCTCCAGAGAC AATTC C AAG AAC AC CCT GT AT CTT C AAAT G AAC AGC CT GAG AGCT G AGG AC ACTGCT GT GT ATT ACT GT GCT AG AGGCT AC GAT G ACT AT GAT GAG AT G AC CT ACTTT AAC AT C TGGGGCCAAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT 142 2016273912 14 Dec 2016

GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCT

C AGC AGCGT GGT GACCGT GCCCTCC AGC AGCTT GGGC ACCC AGACCTAC ATCTGC A

ACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT

T GTGAC AAAACT C AC AC AT GCCC ACCGT GCCC AGC ACCT GAACTCCTGGGGGGACC

GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC

TGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA

ACT GGT ACGT GGACGGCGT GGAGGT GC AT AAT GCC AAGAC AAAGCCGCGGGAGGA

GCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG

GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA

TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC

CTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT

CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG

AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT

AC AGC AAGCT C ACCGT GGAC AAGAGC AGGT GGC AGC AGGGGAACGT CTT CT C ATGC

TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGTAAATGA (SEQ ID NO: 214).

[00587] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 215; SEQ ID NO: 216; and SEQ ID NO: 217 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12.

[00588] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 218; SEQ ID NO: 219; and SEQ ID NO: 220 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14. 143 2016273912 14 Dec 2016 [00589] The invention also optional contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 211 encoding the light chain variable sequence of SEQ ID NO: 11; the polynucleotide SEQ ID NO: 212 encoding the light chain sequence of SEQ ID NO: 12; the polynucleotide SEQ ID NO: 213 encoding the heavy chain variable sequence of SEQ ID NO: 13; the polynucleotide SEQ ID NO: 214 encoding the heavy chain sequence of SEQ ID NO: 14; polynucleotides encoding the complementaritydetermining regions (SEQ ID NO: 215; SEQ ID NO: 216; and SEQ ID NO: 217) of the light chain variable sequence of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 218; SEQ ID NO: 219; and SEQ ID NO: 220) of the heavy chain variable sequence of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14.

[00590] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments for treatment or prevention of pain and pain associated conditions having binding specificity for NGF. With respect to antibody Ab2, the polynucleotides encoding the full length Ab2 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 212 encoding the light chain sequence of SEQ ID NO: 12 and the polynucleotide SEQ ID NO: 214 encoding the heavy chain sequence of SEQ ID NO: 14.

[00591] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab2 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Ab2 or Fab fragments thereof may be produced via expression of Ab2 polynucleotides in mammalian 144 2016273912 14 Dec 2016 cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00592] Antibody Ab3 [00593] The invention is further directed to the use of polynucleotides set forth below to produce antibody Ab3 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Ab3 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 21: [00594] GCAGCCGTGCTGACCCAGACACCATCGCCCGTGTCTGCAGCTATGGGA G AC AC AGT C AC CAT C AAGT GCC AGTCC AGT C AG AGT GTTT AT AAG AAC AACTACTT A TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAGGCTCCTGATCTATGATGCATCC AAT CT GCC ATCTGGGGT CCC AT C ACGGTT C AGCGGC AGT GG AT CT GGG AC AC AGTT C ACT CT C AC CAT C AGCGGCGTGC AGT GT G ACG AT GCTGCC ACTT ACT ACT GT CT AGGC GATT AT GAT GAT GAT GCT GAT AAT GCTTT CGGCGG AGGG ACCG AGGTGGT GGT C AA ACGT (SEQ ID NO: 221).

[00595] In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 22:

[00596] GCAGCCGTGCTGACCCAGACACCATCGCCCGTGTCTGCAGCTATGGGA G AC AC AGT C ACC AT C AAGT GCC AGTCC AGT C AGAGT GTTT AT AAGAAC AACTACTT A TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAGGCTCCTGATCTATGATGCATCC AAT CT GCC ATCTGGGGT CCC AT C ACGGTT C AGCGGC AGT GGAT CT GGG AC AC AGTT C ACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTT ACT ACTGTCT AGGC 145 2016273912 14 Dec 2016 GATTATGATGATGATGCTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGT AC AGT GGAAGGT GGATAACGCCCT CC AAT CGGGT AACT CCC AGG AG AGT GTC A C AG AGC AGGAC AGC AAGGAC AGC ACCT AC AGCCT C AGC AGC ACCCT G ACGCT G AGC AAAGC AG ACT ACGAGAAAC AC AAAGT CT ACGCCTGCGAAGT C ACCC AT C AGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 222).

[00597] In another embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 23: [00598] C AGT C GGT GG AGG AGTCCGGGGGT CGCCT GGT C ACGCCT GGG AC ACC CCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGTAATGATCTGG GTCCGC C AGGCTC C AGGG A AGGGGCT GGAAT AC AT CGG AAT C ACTT GG AGT GCTGG TACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGA CCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTC TGTGCCGGAGGTGGTGGTAGTATTTATGATATTTGGGGCCCGGGCACCCTGGTCACC GTCTCGAGC (SEQ ID NO: 223).

[00599] In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 24:

[00600] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGTAATGATCTGG GT CCGCC AGGCTCC AGGGAAGGGGCT GGAAT AC AT CGGAAT C ACTT GGAGT GCTGG TACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGA CCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTC TGTGCCGGAGGTGGTGGTAGTATTTATGATATTTGGGGCCCGGGCACCCTGGTCACC GTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC 146 2016273912 14 Dec 2016

CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT

CC AGC AGCTT GGGC ACCC AG ACCT AC AT CT GC AACGT GAATC AC AAGCCC AGC AAC

ACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCC

ACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA

ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGG

ACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG

GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGT

GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT

GCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC

AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT

GACC AAGAACC AGGT C AGCCTGACCT GCCTGGTC AAAGGCTT CT ATCCC AGCGAC A

TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT

CCCGT GCT GGACTCCGACGGCTCCTT CTTCCT CT AC AGC AAGCTC ACCGT GG AC AAG

AGC AGGT GGC AGC AGGGG AACGT CTTCTC AT GCTC CGT GAT GC AT G AGGCT CT GC AC AAC C ACT AC ACGC AG A AG AGCCT CTCC CT GT CTCC GGGT AAAT G A (SEQ ID NO: 224).

[00601] In a further embodiment of the invention, polynucleotides encoding antibody fragments having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 225; SEQ ID NO: 226; and SEQ ID NO: 227 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22.

[00602] In a further embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 228; SEQ ID NO: 229; and SEQ ID NO: 230 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24. 147 2016273912 14 Dec 2016 [00603] The invention also contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments described herein for treatment or prevention of pain and pain associated conditions. In one embodiment of the invention, polynucleotides encoding antibody fragments having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 221 encoding the light chain variable sequence of SEQ ID NO: 21; the polynucleotide SEQ ID NO: 222 encoding the light chain sequence of SEQ ID NO: 22; the polynucleotide SEQ ID NO: 223 encoding the heavy chain variable sequence of SEQ ID NO: 23; the polynucleotide SEQ ID NO: 224 encoding the heavy chain sequence of SEQ ID NO: 24; polynucleotides encoding the complementaritydetermining regions (SEQ ID NO: 225; SEQ ID NO: 226; and SEQ ID NO: 227) of the light chain variable sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 228; SEQ ID NO: 229; and SEQ ID NO: 230) of the heavy chain variable sequence of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24.

[00604] In a preferred embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments for treatment or prevention of pain and pain associated conditions having binding specificity for NGF. With respect to antibody Ab3, the polynucleotides encoding the full length Ab3 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 222 encoding the light chain sequence of SEQ ID NO: 22 and the polynucleotide SEQ ID NO: 224 encoding the heavy chain sequence of SEQ ID NO: 24.

[00605] Another embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab3 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Ab3 or Fab fragments thereof may be produced via expression of Ab3 polynucleotides in mammalian 148 2016273912 14 Dec 2016 cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00606] Antibody Ab4 [00607] The invention is further directed to the use of polynucleotides set forth below to produce antibody Ab4 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Ab4 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 31: [00608] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA G AC AG AGT C ACC AT C ACTT GCC AGT CC AGTC AG AAT GTTT AT AAG AAC AACT ACTTA TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC ACT CT GGC AT CTGGGGTCCC ATCTCGTTTC AGT GGC AGT GG ATCTGGG AC AGATTTC ACT CT C ACC AT C AGC AGCCT GC AGCCT G AAG AT GTT GC AACTT ATT ACT GT GC AGGC GGTTAT ACC AGT AGT AGT GAT AATGCTTTCGGCGG AGG AACC AAGGT GG AAATC AA ACGT (SEQ ID NO: 231).

[00609] In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 32:

[00610] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA G AC AG AGT C ACC AT C ACTT GCC AGT CC AGTC AG AATGTTT AT AAG AAC AACT ACTTA TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC ACT CT C ACC AT C AGC AGCCT GC AGCCT GAAG AT GTTGC AACTT ATT ACT GT GC AGGC 149 2016273912 14 Dec 2016 GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA AT CT GG AACT GCCT CT GTT GT GT GCCTGCT G AATAACTT CT ATCCC AG AG AGGCC AA AGT AC AGT GGAAGGT GGAT A ACGCCCT CC AAT CGGGT AACT CCC AGG AG AGT GTC A C AG AGC AGGAC AGC AAGG AC AGC ACCT AC AGCCT C AGC AGC ACCCT G ACGCT G AGC AAAGC AGACT ACGAGAAAC AC AAAGT CT ACGCCTGCGAAGT C ACCC AT C AGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 232).

[00611] In another embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 33: [00612] G AGGT GC AGCT GGT GG AGT CT GGGGGAGGCTT GGT CC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCTATGTAATGAT CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAATCACTTGGAGTG CTGGTACATACTACGCGAGCAGTGCGAAAGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCTGTG T ATT ACT GT GCTGG AGGT GGT GGT AGT AT CT AT GAT ATTT GGGGCC AAGGGACCCT C GTCACCGTCTCGAGC (SEQ ID NO: 233).

[00613] In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 34:

[00614] GAGGT GC AGCTGGT GGAGTCT GGGGGAGGCTT GGTCC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCTATGTAATGAT CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAATCACTTGGAGTG CTGGTAC ATACT ACGCG AGC AGT GCGAAAGGCCGATT C ACC AT CTCC AGAGAC AAT TCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCTGTG T ATT ACT GT GCTGG AGGT GGT GGT AGT AT CT AT GAT ATTT GGGGC C AAGGG AC CCTC GTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTT CCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACA 150 2016273912 14 Dec 2016 CCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA GC AAC ACC AAGGT GG AC AAG AG AGTT G AGCC C AAAT CTT GT G AC AAAACT C AC AC A TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGT GG ACGT G AGC C ACG AAG ACC CT G AGGT C AAGTT C AACT GGT ACGT GGACGGCG TGG AGGT GC AT AAT GC C AAG AC AAAGCCGCGGG AGGAGC AGT ACGCC AGC ACGT A CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT GG AC A AG AGC AGGT GGC AGC AGGGG AACGTCTT CT CAT GCT CCGTGAT GC AT GAGG CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO: 234).

[00615] In a further embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 235; SEQ ID NO: 236; and SEQ ID NO: 237 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32.

[00616] In a further embodiment of the invention, polynucleotides encoding antibody fragments h for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 238; SEQ ID NO: 239; and SEQ ID NO: 240 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34. 151 2016273912 14 Dec 2016 [00617] The invention also contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 231 encoding the light chain variable sequence of SEQ ID NO: 31; the polynucleotide SEQ ID NO: 232 encoding the light chain sequence of SEQ ID NO: 32; the polynucleotide SEQ ID NO: 233 encoding the heavy chain variable sequence of SEQ ID NO: 33; the polynucleotide SEQ ID NO: 234 encoding the heavy chain sequence of SEQ ID NO: 34; polynucleotides encoding the complementaritydetermining regions (SEQ ID NO: 235; SEQ ID NO: 236; and SEQ ID NO: 237) of the light chain variable sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 238; SEQ ID NO: 239; and SEQ ID NO: 240) of the heavy chain variable sequence of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34.

[00618] In a preferred embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments for treatment or prevention of pain and pain associated conditions having binding specificity for NGF. With respect to antibody Ab4, the polynucleotides encoding the full length Ab4 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 232 encoding the light chain sequence of SEQ ID NO: 32 and the polynucleotide SEQ ID NO: 234 encoding the heavy chain sequence of SEQ ID NO: 34.

[00619] Another embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab4 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies for treatment or prevention of pain and pain associated conditions such as Ab4 or Fab fragments thereof may be 152 2016273912 14 Dec 2016 produced via expression of Ab4 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00620] Antibody Ab5 [00621] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Ab5 polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Ab5 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 41: [00622] GCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGA GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTTACAGCAATTTAGCCTG GTATCAGCAGAGACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCCACTCT GGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTC TCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCTCTTACTACTGTCAACAGGGTT TTACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC GT (SEQ ID NO: 241).

[00623] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 42:

[00624] GCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGA GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTTACAGCAATTTAGCCTG GTATCAGCAGAGACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCCACTCT 153 2016273912 14 Dec 2016

GGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTC

TCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCTCTTACTACTGTCAACAGGGTT

TTACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAC

GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT

CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG

T AC AGT GGAAGGT GGAT AACGCCCT CC AAT CGGGT AACT CCC AGG AG AGTGT C AC A

GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA

AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 242).

[00625] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 43: [00626] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAACTATGCAGTGGGCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGGTCGTAATG GTAACACATGGTACGCGAGCTGGGCAAGAGGCCGATTCACCATCTCCAAAACCTCG ACCACGGTGGATCTGAAAATCACCAGTCCGACAAGCGAGGACACGGCCACATATTT CTGTGCCAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCTGGGGCCC AGGCACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 243).

[00627] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 44:

[00628] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAACTATGCAGTGGGCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGGTCGTAATG GTAACACATGGTACGCGAGCTGGGCAAGAGGCCGATTCACCATCTCCAAAACCTCG ACCACGGTGGATCTGAAAATCACCAGTCCGACAAGCGAGGACACGGCCACATATTT CTGTGCCAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCTGGGGCCC AGGCACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCT 154 2016273912 14 Dec 2016

GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA

AGGACTACTTCCCCGAACCGGT GACGGTGTCGT GGAACT C AGGCGCCCT G ACC AGC

GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC

GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT

CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAA

AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT

CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC

ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG

TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGC

C AGC ACGT ACC GT GT GGT C AGC GT C CT C ACC GT C CT GC AC C AGG ACT GGCTG AAT GG

CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA

CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA

TCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT

CT ATCCC AGCGAC ATCGCCGT GGAGT GGGAG AGC AAT GGGC AGCCGGAGAAC AACT

ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC

TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG

CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA TGA (SEQ ID NO: 244).

[00629] In a further optional embodiment of the invention, polynucleotides encoding for treatment or prevention of pain and pain associated conditions fragments having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 245; SEQ ID NO: 246; and SEQ ID NO: 247 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42.

[00630] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 248; SEQ ID NO: 249; and SEQ ID NO: 250 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or 155 2016273912 14 Dec 2016 hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.

[00631] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 241 encoding the light chain variable sequence of SEQ ID NO: 41; the polynucleotide SEQ ID NO: 242 encoding the light chain sequence of SEQ ID NO: 42; the polynucleotide SEQ ID NO: 243 encoding the heavy chain variable sequence of SEQ ID NO: 43; the polynucleotide SEQ ID NO: 244 encoding the heavy chain sequence of SEQ ID NO: 44; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 245; SEQ ID NO: 246; and SEQ ID NO: 247) of the light chain variable sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 248; SEQ ID NO: 249; and SEQ ID NO: 250) of the heavy chain variable sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.

[00632] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab5, the polynucleotides encoding the full length Ab5 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 242 encoding the light chain sequence of SEQ ID NO: 42 and the polynucleotide SEQ ID NO: 244 encoding the heavy chain sequence of SEQ ID NO: 44.

[00633] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention described herein (infra), Fab fragments may be produced by 156 2016273912 14 Dec 2016 enzymatic digestion (e.g., papain) of Ab5 following expression of the full-length polynucleotides in a suitable host. In another optional embodiment of the invention, anti-NGF antibodies such as Ab5 or Fab fragments thereof may be produced via expression of Ab5 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00634] Antibody Ab6 [00635] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Ab6 polypeptides having binding specificity to NGF, which inhibits the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Ab6 polypeptides.

The invention is further directed to polynucleotides encoding for treatment or prevention of pain and pain associated conditions polypeptides having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 51: [00636] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG G AAT CT GGAGT CC C AT C AAGGTT C AGCGGC AGT GGAT CT GGGAC AGAGT AC ACTCT CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAACAGGGTTT TACT GTT AGTGAT ATT GAT AAT GCTTT CGGCGG AGG AACC AAGGT GG A AATC AAAC GT (SEQ ID NO: 251).

[00637] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 52:

[00638] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG 157 2016273912 14 Dec 2016 TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG G AAT CT GGAGT CCC AT C AAGGTTC AGCGGC AGT GGAT CT GGGAC AG AGT AC ACT CT C ACC AT C AGC AGCCT GC AGCCT GAT G ATTTTGC AACTT ACT ACT GCC AAC AGGGTTT T ACT GTT AGTGATATT GAT AAT GCTTT CGGCGG AGG AACC AAGGT GG A AATC AAAC GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG T AC AGT GGAAGGT GGAT AACGCCCT CC AAT CGGGT AACT CCC AGG AG AGT GT C AC A GAGC AGGACAGC AAGG AC AGC ACCT AC AGCCTC AGC AGC ACCCT G ACGCT G AGC AA AGC AGACT ACGAGAAAC AC AAAGTCT ACGCCT GCG AAGT C ACCC AT C AGGGCCT G A GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 252).

[00639] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 53: [00640] GAGGT GC AGCT GGT GGAGT CT GGGGG AGGCTT GGT CC AGCCTGGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC AATTC C AAG AAC AC CCT GT AT CTT C AAAT GAAC AGCCT GAGAGCT G AGG AC ACTGCT GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCT GGGGCCCAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 253).

[00641] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 54:

[00642] GAGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGTCC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG CT GGGTCCGTC AGGCTCC AGGGAAGGGGCTGGAGT GGGTCGGAATC ATT GGTCGTA ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC AATT C C AAG AAC AC CCT GT AT CTT C AAAT GAAC AGC CTG AG AGCT G AGG AC ACTGCT GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCT 158 2016273912 14 Dec 2016

GGGGCCCAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCT

TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC

TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG

ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC

AGC AGCGT GGT GACCGTGCCCTCC AGC AGCTT GGGC ACCC AGACCT AC ATCT GC AA

CGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTT

GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT

GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA

CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC

AGT ACGCC AGC ACGT ACCGT GT GGTC AGCGT CCTC ACCGT CCT GC ACC AGG ACT GGC

T GAATGGC AAGGAGT AC AAGT GC AAGGT CT CC AAC AAAGCCCT CCC AGCCCCC AT C

GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT

GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA

AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG

AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGC AAGCT C AC CGT GG AC AAG AGC AGGT GGC AGC AGGGGAACGT CTT CT CAT GCT C

CGT GAT GC AT GAGGCTCT GC AC AACC ACT AC ACGC AGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA (SEQ ID NO: 254).

[00643] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52.

[00644] In a further optional embodiment of the invention, polynucleotides encoding for treatment or prevention of pain and pain associated conditions fragments having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260 which correspond to 159 2016273912 14 Dec 2016 polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54.

[00645] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 251 encoding the light chain variable sequence of SEQ ID NO: 51; the polynucleotide SEQ ID NO: 252 encoding the light chain sequence of SEQ ID NO: 52; the polynucleotide SEQ ID NO: 253 encoding the heavy chain variable sequence of SEQ ID NO: 53; the polynucleotide SEQ ID NO: 254 encoding the heavy chain sequence of SEQ ID NO: 54; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257) of the light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260) of the heavy chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54.

[00646] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab6, the polynucleotides encoding the full length Ab6 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 252 encoding the light chain sequence of SEQ ID NO: 52 and the polynucleotide SEQ ID NO: 254 encoding the heavy chain sequence of SEQ ID NO: 54.

[00647] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. 160 2016273912 14 Dec 2016

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab6 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Ab6 or Fab fragments thereof may be produced via expression of Ab6 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00648] Antibody Ab7 [00649] The invention optionally is further directed to the use of polynucleotides set forth below to produce antibody Ab7 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75. in methods of treating pain in an individual comprising administering to said individual antibody Ab7 polypeptides.

The invention is further directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 61: [00650] GCCGATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTCAACCTGTG GGAGGC AC AGT C ACC AT C AAGT GCC AGGCC AGT G AGGAC ATTTAT AACTT ATT GGC CT GGT ATC AGC AGAAACC AGGGC AGCCT CCC AAGCT CCT GAT CT ATT CT GC AT CC AC TCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACA CT CT C ACC AT C AGCGGCCT GGAGT GT GCCG AT GCT GCC ACTT ACT ACTGT C AAAAC A ATTATCTTGTTACTACTTATGGTGTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCA AACGT (SEQ ID NO: 261).

[00651] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 62: 161 2016273912 14 Dec 2016 [00652] GCCGATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTCAACCTGTG GGAGGC AC AGT C ACC AT C AAGT GCC AGGCC AGT GAGGAC ATTTAT AACTT ATT GGC CT GGT ATC AGC AGAAACC AGGGC AGCCT CCC AAGCT CCT GAT CT ATT CT GC AT CC AC TCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACA CT CT C ACC AT C AGCGGCCT GG AGT GT GCCG AT GCT GCC ACTT ACT ACT GT C AAAAC A ATTATCTTGTTACTACTTATGGTGTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCA AACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAG C AAAGC AGACT ACGAG AAAC AC AAAGT CT ACGCCT GCG AAGT C ACCC ATC AGGGCC T GAGCT CGCCCGT C AC AAAG AGCTT C AAC AGGGG AG AGT GTT AG (SEQ ID NO: 262).

[00653] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 63: [00654] C AGGAGC AGCT GAAGGAGT CC GGGGGT CGC CT GGT C ACGC CT GGGAC ACCCCTGACACTCACCTGTACAGTCTCTGGATTCTCCCTCAGTAGCTATGCAATGAT CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTGATACTG ATACTAGCGCATACTACGCGAGCTGGGTGAAAGGCCGATTCACCATCTCCAGAACC TCGACCACGGTGGATCTCAAAATCACTAGTCCGACAACCGAGGACACGGCCACCTA TTTCT GT GC C AGAT CTTAT GCT GCTTAT GGT GGTT AT CCT GCT ACTTTT G ATCCCT GG GGCCCAGGCACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 263).

[00655] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 64:

[00656] CAGGAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGAC ACCCCTGACACTCACCTGTACAGTCTCTGGATTCTCCCTCAGTAGCTATGCAATGAT CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTGATACTG ATACTAGCGCATACTACGCGAGCTGGGTGAAAGGCCGATTCACCATCTCCAGAACC 162 2016273912 14 Dec 2016

TCGACCACGGTGGATCTCAAAATCACTAGTCCGACAACCGAGGACACGGCCACCTA

TTT CT GTGCC AGAT CTTAT GCT GCTT AT GGT GGTT AT CCTGCT ACTTTT G ATCCCTGG

GGCCCAGGCACCCTGGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTC

CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG

GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC

CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG

C AGCGT GGTG ACCGT GCCCT CC AGC AGCTT GGGC ACCC AG ACCT AC AT CT GC AACGT

GAAT C AC AAGCCC AGC AAC ACC AAGGT GGAC AAG AG AGTT G AGCCC AAATCTT GT G

AC AAAACT C AC AC AT GCCCACCGTGCCC AGC ACCT G AACT CCT GGGGGG ACCGT C A

GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG

GT C AC AT GCGTGGT GGT GGACGTGAGCC ACGAAG ACCCT G AGGTC AAGTT C AACT G

GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG

TACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG

AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA

GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC

CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA

GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA

CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG

C AAGCT C AC CGT GGAC AAG AGC AGGT GGC AGC AGGGGAACGT CTT CT CAT GCTCCG

TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGA (SEQ ID NO: 264).

[00657] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 265; SEQ ID NO: 266; and SEQ ID NO: 267 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62.

[00658] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having 163 2016273912 14 Dec 2016 binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 268; SEQ ID NO: 269; and SEQ ID NO: 270 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64.

[00659] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 261 encoding the light chain variable sequence of SEQ ID NO: 61; the polynucleotide SEQ ID NO: 262 encoding the light chain sequence of SEQ ID NO: 62; the polynucleotide SEQ ID NO: 263 encoding the heavy chain variable sequence of SEQ ID NO: 63; the polynucleotide SEQ ID NO: 264 encoding the heavy chain sequence of SEQ ID NO: 64; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 265; SEQ ID NO: 266; and SEQ ID NO: 267) of the light chain variable sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 268; SEQ ID NO: 269; and SEQ ID NO: 270) of the heavy chain variable sequence of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64.

[00660] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab7, the polynucleotides encoding the full length Ab7 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 262 encoding the light chain sequence of SEQ ID NO: 62 and the polynucleotide SEQ ID NO: 264 encoding the heavy chain sequence of SEQ ID NO: 64.

[00661] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such 164 2016273912 14 Dec 2016 as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab7 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Ab7 or Fab fragments thereof may be produced via expression of Ab7 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00662] Antibody Ab8 [00663] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Ab8 polypeptides having binding specificity to NGF, which inhibits the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Ab8 polypeptides.

The invention is further optionally directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 71: [00664] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTGAGGACATTTACAACTTATTGGCCTGG T AT C AGC AG AAAC C AGGGAAAGT CCCT AAGCT CCT G ATCT ATTCT GC AT CC ACTCT G GCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTC ACC AT C AGC AGC CT GC AGCCT GAAGAT GTT GC AACTT ATT ACT GT C AAAAC AACT AT CTT GTT ACT ACTT AT GGTGTT GCTTT CGGCGG AGG AACC AAGGT GGAAATC AAACGT (SEQ ID NO: 271).

[00665] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 72: 165 2016273912 14 Dec 2016 [00666] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTGAGGACATTTACAACTTATTGGCCTGG TATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATTCTGCATCCACTCTG GC AT CT GGGGT CCC AT CT CGTTT C AGT GGC AGTGGATCT GGGAC AGATT AC ACTCT C ACC ATC AGC AGCCT GC AGCCT GAAGAT GTT GC AACTT ATT ACTGT C AAAAC AACT AT CTTGTTACTACTTATGGTGTTGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGT ACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGC TCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 272).

[00667] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 73: [00668] C AGGT AC AGCT GGT GG AGT CT GGT GGAGGCGT GGT CC AGCCT GGGAG GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCACCTTCAGTAGCTATGCAATGATC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTGATACTGA TACT AGCGC AT ACT AC GC AAGC AGT GT G AAAGGC CG ATT C ACC AT CT CC AGAGAC A ATTCC AAGAAC ACGCT GT ACCT GC AAAT GT CT AGCCTGAGAGCCGAGGAC ACGGCT GTGTATTACTGTGCTAGATCTTATGCTGCTTATGGTGGTTATCCTGCTACTTTTGATC CCTGGGGCCAAGGTACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 273).

[00669] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 74:

[00670] C AGGT AC AGCT GGTGG AGTCT GGT GGAGGCGT GGTCC AGCCT GGGAG GTCCCTGAGACT CTCCTGT GC AGCTT CT GGATTC ACCTTC AGTAGCT ATGC AATG ATC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTGATACTGA TACTAGCGCATACTACGCAAGCAGTGTGAAAGGCCGATTCACCATCTCCAGAGACA 166 2016273912 14 Dec 2016

ATTCC AAGAAC ACGCT GT ACCT GC AAAT GT CT AGCCTGAGAGCCGAGGAC ACGGCT

GTGTATTACTGTGCTAGATCTTATGCTGCTTATGGTGGTTATCCTGCTACTTTTGATC

CCTGGGGCCAAGGTACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGG

TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT

GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC

CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC

CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC

AAC GT G AAT C AC AAGCC C AGC AAC AC C AAGGT GG AC AAG AG AGTT G AGCCC AAAT C

TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAC

CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC

CT G AGGT C AC AT GC GT GGT GGTGG ACGT G AGCC ACG AAG ACCCT G AGGTC AAGTT C

AACT GGT ACGT GGACGGCGT GGAGGT GC ATAAT GCC AAG AC A AAGCCGCGGG AGG

AGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT

GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCC

ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC

CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG

TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG

GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC

TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG

CT CCGT GAT GC AT G AGGCT CT GC AC AACC ACT AC ACGC AGAAGAGCCT CTCCCT GT C TCCGGGTAAATGA (SEQ ID NO: 274).

[00671] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 275; SEQ ID NO: 276; and SEQ ID NO: 277 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72.

[00672] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having 167 2016273912 14 Dec 2016 binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 278; SEQ ID NO: 279; and SEQ ID NO: 280 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74.

[00673] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 271 encoding the light chain variable sequence of SEQ ID NO: 71; the polynucleotide SEQ ID NO: 272 encoding the light chain sequence of SEQ ID NO: 72; the polynucleotide SEQ ID NO: 273 encoding the heavy chain variable sequence of SEQ ID NO: 73; the polynucleotide SEQ ID NO: 274 encoding the heavy chain sequence of SEQ ID NO: 74; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 275; SEQ ID NO: 276; and SEQ ID NO: 277) of the light chain variable sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 278; SEQ ID NO: 279; and SEQ ID NO: 280) of the heavy chain variable sequence of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74.

[00674] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab8, the polynucleotides encoding the full length Ab8 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 272 encoding the light chain sequence of SEQ ID NO: 72 and the polynucleotide SEQ ID NO: 274 encoding the heavy chain sequence of SEQ ID NO: 74.

[00675] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such 168 2016273912 14 Dec 2016 as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab8 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Ab8 or Fab fragments thereof may be produced via expression of Ab8 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00676] Antibody Ab9 [00677] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Ab9 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Ab9 polypeptides.

The invention is further optionally directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 81: [00678] GCCTATGATATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGA GGC AC AGT C ACC AT C AAGT GC C AGGC C AGT GAGAAC ATT GGT AGCT ACTT AGCCT G GTATCAGCAGAAACCAGGGCAGCCTCCCGAACTCCTGATCTACAGGGCGTCCACTCT GGC AT CT GGGGTCC C AT C GCGGTT C AAAGGC AGT GG AT CT GGG AC AC AGTT C ACT CT C AC CAT C AGCGGC GT GG AGT GT GC CG AT GCT GCC ACTT ACTACT GT C AAC AGGGTT A T A AT AGT G AG AATCTT GAT AAT GCTTTC GGCGG AGGG ACCGAGGTGGTGGTC AAAC GT (SEQ ID NO: 281).

[00679] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 82: 169 2016273912 14 Dec 2016 [00680] GCCTAT GAT AT G ACCC AG ACT CC AGCCT CCGT GT CTGC AGCT GT GGGA GGCACAGTCACCATCAAGTGCCAGGCCAGTGAGAACATTGGTAGCTACTTAGCCTG GTATCAGCAGAAACCAGGGCAGCCTCCCGAACTCCTGATCTACAGGGCGTCCACTCT GGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCT C ACC AT C AGCGGCGT GG AGT GT GCCG AT GCT GCC ACTT ACT ACT GT C AAC AGGGTT A T A ATAGT GAGAAT CTT GAT AAT GCTTTCGGCGG AGGG ACCG AGGT GGT GGT C AAAC GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG T AC AGT GGAAGGT GG AT AACGCCCT CC AAT CGGGT AACT CCC AGG AG AGT GT C AC A GAGC AGGACAGC AAGG AC AGC ACCT AC AGCCTC AGC AGC ACCCT G ACGCT G AGC AA AGC AGACT ACGAGAAAC AC AAAGTCT ACGCCT GCG AAGT C ACCC AT C AGGGCCT G A GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 282).

[00681] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 83: [00682] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTATGTATTCAATGGGCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATGGATTAGTTATGGTG GTACTGCATATTACGCGAGCTGGGCGAAGGGCCGATTCACCATCTCCAAAACCTCG ACCACGGTGGAGCTGAAGATCACCAGTCCGACAATCGAGGACACGGCCACCTATTT CTGTGCCAGAGAGACTCCTGTTAATTATTATTTGGACATTTGGGGCCAGGGGACCCT CGTCACCGTCTCGAGC (SEQ ID NO: 283).

[00683] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 84:

[00684] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTATGTATTCAATGGGCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATGGATTAGTTATGGTG GTACTGCATATTACGCGAGCTGGGCGAAGGGCCGATTCACCATCTCCAAAACCTCG 170 2016273912 14 Dec 2016

ACCACGGTGGAGCTGAAGATCACCAGTCCGACAATCGAGGACACGGCCACCTATTT

CTGTGCCAGAGAGACTCCTGTTAATTATTATTTGGACATTTGGGGCCAGGGGACCCT

CGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC

CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT

TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC

ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC

GTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC

AGC AAC AC C AAGGT GGAC AAG AGAGTTGAGCCC AAATCTT GT GAC AAAACT C AC AC

ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC

CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT

GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTA

CC GTGT GGT C AGCGT CCT C ACCGT CCTGC ACC AGGACT GGCT GAAT GGC AAGGAGT

ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC

AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA

GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA

GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC

CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT

GG AC A AG AGC AGGT GGC AGC AGGGG AACGTCTT CT CAT GCT CCGT GAT GC AT GAGG

CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO: 284).

[00685] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 285; SEQ ID NO: 286; and SEQ ID NO: 287 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82.

[00686] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having 171 2016273912 14 Dec 2016 binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 288; SEQ ID NO: 289; and SEQ ID NO: 290 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84.

[00687] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 281 encoding the light chain variable sequence of SEQ ID NO: 81; the polynucleotide SEQ ID NO: 282 encoding the light chain sequence of SEQ ID NO: 82; the polynucleotide SEQ ID NO: 283 encoding the heavy chain variable sequence of SEQ ID NO: 83; the polynucleotide SEQ ID NO: 284 encoding the heavy chain sequence of SEQ ID NO: 84; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 285; SEQ ID NO: 286; and SEQ ID NO: 287) of the light chain variable sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 288; SEQ ID NO: 289; and SEQ ID NO: 290) of the heavy chain variable sequence of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84.

[00688] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab9, the polynucleotides encoding the full length Ab9 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 282 encoding the light chain sequence of SEQ ID NO: 82 and the polynucleotide SEQ ID NO: 284 encoding the heavy chain sequence of SEQ ID NO: 84.

[00689] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such 172 2016273912 14 Dec 2016 as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab9 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Ab9 or Fab fragments thereof may be produced via expression of Ab9 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00690] Antibody AblO

[00691] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody AblO polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody AblO polypeptides. The invention is further optionally directed to polynucleotides encoding antibody polypeptides having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 91: [00692] GCCTATGATATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG ACAGAGTCACCATCACTTGCCAGGCCAGTGAGAACATTGGTAGCTACTTAGCCTGGT ATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAGGGCTTCCACTCTGG CATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAACAGGGTTACA ATAGTGAGAATCTTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 291).

[00693] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 92: 173 2016273912 14 Dec 2016 [00694] GCCTATGATATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG ACAGAGTCACCATCACTTGCCAGGCCAGTGAGAACATTGGTAGCTACTTAGCCTGGT ATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAGGGCTTCCACTCTGG CATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CC ATC AGC AGC CT GC AGC CT G AAG AT GTT GC AACTT ATT ACT GT C AAC AGGGTTAC A AT AGTGAGAATCTT GATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATC AAACGT ACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGC TCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 292).

[00695] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 93: [00696] C AGGT AC AGCTGGTGG AGTCTGGT GG AGGCGT GGTCC AGCCT GGG AG GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCACCTTCAGTATGTATTCAATGGGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATGGATTAGTTATGG TGGTACTGCATACTACGCTAGCAGCGCTAAGGGCCGATTCACCATCTCCAGAGACA ATTCC AAGAAC ACGCT GT ACCT GC AAAT GT CT AGCCTGAGAGCCGAGGAC ACGGCT GT GT ATT ACT GT GCT AG AG AG ACTC CT GTT AATT ACT ACTT GG AC ATTT GGGGCC AA GGTACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 293).

[00697] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 94:

[00698] CAGGTACAGCTGGTGGAGTCTGGTGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCACCTTCAGTATGTATTCAATGGGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATGGATTAGTTATGG TGGTACTGCATACTACGCTAGCAGCGCTAAGGGCCGATTCACCATCTCCAGAGACA 174 2016273912 14 Dec 2016

ATTCC AAGAAC ACGCT GT ACCT GC AAAT GT CT AGCCTGAGAGCCGAGGAC ACGGCT

GTGTATTACTGTGCTAGAGAGACTCCTGTTAATTACTACTTGGACATTTGGGGCCAA

GGTACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTG

GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAA

GGACT ACTTCCCCGAACCGGT GACGGT GTCGT GGAACT C AGGCGCCCT GACC AGCG

GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG

TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC

AC A AGCC C AGC A AC AC C AAGGT GG AC AAG AG AGTT G AGC CC AAATCTT GT G AC AAA

ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC

CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA

TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT

GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCC

AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC

AAGGAGT AC AAGTGC AAGGT CTCC AAC AAAGCCCTCCCAGCCCCC ATCGAGAAAAC

CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT

CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC

T AT C CC AGC G AC AT CGCCGT GG AGT GGG AG AGC AAT GGGC AGCCGGAGA AC AACT A

CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT

CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC

AT GAGGCT CT GC AC A ACC ACT AC ACGC AG AAGAGCCT CTCCCT GT CT CCGGGT AAAT GA (SEQ ID NO: 294).

[00699] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 295; SEQ ID NO: 296; and SEQ ID NO: 297 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92.

[00700] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having 175 2016273912 14 Dec 2016 binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 298; SEQ ID NO: 299; and SEQ ID NO: 300 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.

[00701] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 291 encoding the light chain variable sequence of SEQ ID NO: 91; the polynucleotide SEQ ID NO: 292 encoding the light chain sequence of SEQ ID NO: 92; the polynucleotide SEQ ID NO: 293 encoding the heavy chain variable sequence of SEQ ID NO: 93; the polynucleotide SEQ ID NO: 294 encoding the heavy chain sequence of SEQ ID NO: 94; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 295; SEQ ID NO: 296; and SEQ ID NO: 297) of the light chain variable sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 298; SEQ ID NO: 299; and SEQ ID NO: 300) of the heavy chain variable sequence of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.

[00702] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody AblO, the polynucleotides encoding the full length AblO antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 292 encoding the light chain sequence of SEQ ID NO: 92 and the polynucleotide SEQ ID NO: 294 encoding the heavy chain sequence of SEQ ID NO: 94.

[00703] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such 176 2016273912 14 Dec 2016 as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of AblO following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as AblO or Fab fragments thereof may be produced via expression of AblO polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00704] Antibody Abll [00705] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Abl 1 polypeptides having binding specificity to NGF, which inhibits the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl 1 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 101: [00706] GCATTCGAATTGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTGGGA GGC AC AGT C ACC AT C AAGT GCC AGGCC AGTCAG AAC ATTGTTACCAATTTAGCCT GG TATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCACTCTG GC AT CT GGGGT CT C ATCGCGGTT C AAAGGC AGTGG ATCTGGGACACAGTTC ACTCTC ACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTATTTCTGTCAGAGCTATGAT GGTTTTAATAGTGCTGGGTTC GGCGGAGGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 301).

[00707] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 102: 177 2016273912 14 Dec 2016 [00708] GCATTCGAATTGACCCAGACTCCATCCTCCGTGGAGGCAGCTGTGGGA GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAACATTGTTACCAATTTAGCCTGG T AT C AAC AG AAACC AGGGC AGCCT CCC AAGCT CCT GAT CT AT GGT GC AT CC ACTCTG GC AT CT GGGGT CT CAT CGCGGTT C AAAGGC AGTGG ATCT GGG AC AC AGTT C ACTCT C ACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTATTTCTGTCAGAGCTATGAT GGTTTT AAT AGT GCT GGGTT CGGCGG AGGG ACCGAGGT GGT GGT C AAACGT ACGGT AGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAC TGCCTCT GTT GTGTGCCT GCT G AAT AACTTCT ATCCC AG AG AGGCC AAAGT AC AGT G GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGG ACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGAC TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCC CGT C AC AAAG AGCTT C AAC AGGGG AG AGT GTT AG (SEQ ID NO: 302).

[00709] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 103: [00710] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTGGCTACGACATGAGCTG GGTCCGCCAGGCTCCAGGAAAGGGGCTGGAATACATCGGACTCATTAGTTATGATG GT AAC AC AT ACTACGCGACCT GGGCGAA AGGCC GATT C ACC AT CT CC AAA ACCT CG ACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT CTGTGCCAGAAGTCTTTATGCTGGTCCTAATGCTGGTATCGGACCGTTTAACATCTG GGGC C AGGGG ACCCTCGTCACCGT CT CG AGC (SEQ ID NO: 303).

[00711] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 104:

[00712] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTGGCTACGACATGAGCTG GGTCCGCCAGGCTCCAGGAAAGGGGCTGGAATACATCGGACTCATTAGTTATGATG GT AAC AC AT ACTACGCGACCT GGGCGAA AGGCC GATT C ACC AT CT CC AAA ACCT CG 178 2016273912 14 Dec 2016

ACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT

CTGTGCCAGAAGTCTTTATGCTGGTCCTAATGCTGGTATCGGACCGTTTAACATCTG

GGGCCAGGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTT

CCCCCTGGCAcCCTCCTCCaAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT

GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA

CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA

GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC

GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTG

TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT

CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG

AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC

AGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC

T G AAT GGC AAGG AGT AC AAGT GC AAGGT CTCC AAC AAAGCCCT CCC AGCCCCC AT C

GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT

GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA

AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG

AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC

AGC AAGCT C ACCGT GG AC AAG AGC AGGT GGC AGC AGGGGAACGT CTT CT CAT GCT C

CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA (SEQ ID NO: 304).

[00713] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 305; SEQ ID NO: 306; and SEQ ID NO: 307 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102.

[00714] In a further optional embodiment of the invention, polynucleotides encoding for treatment or prevention of pain and pain associated conditions fragments having binding 179 2016273912 14 Dec 2016 specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 308; SEQ ID NO: 309; and SEQ ID NO: 310 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104.

[00715] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 301 encoding the light chain variable sequence of SEQ ID NO: 101; the polynucleotide SEQ ID NO: 302 encoding the light chain sequence of SEQ ID NO: 102; the polynucleotide SEQ ID NO: 303 encoding the heavy chain variable sequence of SEQ ID NO: 103; the polynucleotide SEQ ID NO: 304 encoding the heavy chain sequence of SEQ ID NO: 104; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 305; SEQ ID NO: 306; and SEQ ID NO: 307) of the light chain variable sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 308; SEQ ID NO: 309; and SEQ ID NO: 310) of the heavy chain variable sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104.

[00716] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl 1, the polynucleotides encoding the full length Abl 1 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 302 encoding the light chain sequence of SEQ ID NO: 102 and the polynucleotide SEQ ID NO: 304 encoding the heavy chain sequence of SEQ ID NO: 104.

[00717] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such 180 2016273912 14 Dec 2016 as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl 1 following expression of the full-length polynucleotides in a suitable host. In another optional embodiment of the invention, anti-NGF antibodies such as Abl 1 or Fab fragments thereof may be produced via expression of Abl 1 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00718] Antibody Ab 12 [00719] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Abl2 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl2 polypeptides. The invention is further optionally directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 111: [00720] GCATTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAACATTGTTACCAACTTAGCCTGG TATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATGGTGCATCCACTCTG GC AT CT GGGGTCCC AT CTCGTTT C AGT GGC AGT GGATCTGGGAC AGATTT C ACTCTC ACC AT C AGC AGC CT GC AGC CT G AAG AT GTT GC AACTT ATT ACT GT C AG AGCT ATG AT GGTTTCAATAGTGCTGGTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 311).

[00721] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 112: 181 2016273912 14 Dec 2016 [00722] GC ATT CC AG AT G ACCC AGT CT CC AT CCTCCCT GT CT GC ATCT GT AGG A GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAACATTGTTACCAACTTAGCCTGG TATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATGGTGCATCCACTCTG GCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTC ACC ATC AGC AGCCT GC AGCCT G AAG AT GTT GC AACTT ATT ACT GTC AG AGCT AT GAT GGTTTCAATAGTGCTGGTTTCGGCGGAGGAACCAAGGTGGAAATCAAACGTACGGT AGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAC TGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGG ACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGAC TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCC CGT C AC AAAGAGCTT C AAC AGGGG AG AGT GTT AG (SEQ ID NO: 312).

[00723] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 113: [00724] C AGGT AC AGCT GGTGG AGTCT GGT GGAGGCGT GGT CC AGCCT GGG AG GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCTCCCTCAGTGGCTACGACATGAG CT GGGTCCGT C AGGCT C C AGGC AAGGGACT GGAGT GGGT GGG ACTC ATT AGTT AT G ATGGTAACACATACTACGCGACCTCCGCGAAAGGCCGATTCACCATCTCCAGAGAC AATTCCAAGAACACGCTGTACCTGCAAATGTCTAGCCTGAGAGCCGAGGACACGGC TGTGTATTACTGTGCTAGAAGTCTTTATGCTGGTCCTAATGCTGGTATCGGACCGTTT AACATCTGGGGCCAAGGTACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 313).

[00725] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 114:

[00726] C AGGT AC AGCT GGTGG AGTCT GGT GGAGGCGT GGT CC AGCCT GGGAG GTCCCTGAGACTCTCCTGTGCAGCTTCTGGATTCTCCCTCAGTGGCTACGACATGAG CTGGGTCCGTCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGGACTCATTAGTTATG ATGGTAACACATACTACGCGACCTCCGCGAAAGGCCGATTCACCATCTCCAGAGAC 182 2016273912 14 Dec 2016

AATTCCAAGAACACGCTGTACCTGCAAATGTCTAGCCTGAGAGCCGAGGACACGGC

TGTGTATTACTGTGCTAGAAGTCTTTATGCTGGTCCTAATGCTGGTATCGGACCGTTT

AACATCTGGGGCCAAGGTACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCC

ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCT

GGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG

GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT

ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA

TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCC

AAAT CTT GT G AC AAA ACT C AC AC AT GCCC ACCGT GCCC AGC ACCT G AACT CCTGGGG

GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG

ACCCCT GAGGT C AC AT GCGT GGTGGTGGACGTG AGCC ACG AAG ACCCT G AGGTC AA

GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG

AGG AGC AGT AC GCC AGC AC GT ACCGT GT GGTC AGCGT CCT C ACCGT CCT GC ACC AG

GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGC

CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT

ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC

CTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA

GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT

CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT

CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAATGA (SEQ ID NO: 314).

[00727] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 315; SEQ ID NO: 316; and SEQ ID NO: 317 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 112.

[00728] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having 183 2016273912 14 Dec 2016 binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 318; SEQ ID NO: 319; and SEQ ID NO: 320 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114.

[00729] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 311 encoding the light chain variable sequence of SEQ ID NO: 111; the polynucleotide SEQ ID NO: 312 encoding the light chain sequence of SEQ ID NO: 112; the polynucleotide SEQ ID NO: 313 encoding the heavy chain variable sequence of SEQ ID NO: 113; the polynucleotide SEQ ID NO: 314 encoding the heavy chain sequence of SEQ ID NO: 114; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 315; SEQ ID NO: 316; and SEQ ID NO: 317) of the light chain variable sequence of SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 112; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 318; SEQ ID NO: 319; and SEQ ID NO: 320) of the heavy chain variable sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114.

[00730] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl2, the polynucleotides encoding the full length Abl2 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 312 encoding the light chain sequence of SEQ ID NO: 112 and the polynucleotide SEQ ID NO: 314 encoding the heavy chain sequence of SEQ ID NO: 114.

[00731] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such 184 2016273912 14 Dec 2016 as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl2 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl2 or Fab fragments thereof may be produced via expression of Abl2 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00732] Antibody Abl 3 [00733] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Abl3 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl3 polypeptides. The invention is further optionally directed to polynucleotides encoding antibody polypeptides having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 121: [00734] GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGA GGC AC AGT C AGC AT C AGTT GCC AGT C C AGT C AGAAT GTTT AT AAGAAC AACT ACTT A TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCC ACT CT GGC AT CT GGGGT CCC AT CGCGGTT C AAAGGCGGT GGATCT GGG AC AG ATTT C ACT CT C ACC AT C AGCGACGT GC AGT GT GACGCT GCT GCC ACTT ACT ACT GT GC AGGC GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA ACGT (SEQ ID NO: 321).

[00735] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 122: 185 2016273912 14 Dec 2016 [00736] GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGA GGC AC AGT C AGC AT C AGTT GCC AGTCC AGT C AGAAT GTTT AT AAGAAC AACTACTT A TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCC ACT CT GGC AT CT GGGGT CCC AT CGCGGTT C AAAGGCGGT GGATCTGGGAC AGATTT C ACT CT C AC CAT C AGCGACGT GC AGT GT G ACGCT GCT GCC ACTTACT ACT GTGC AGGC GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGT AC AGT GG AAGGT GG AT A ACGCCCT CC AAT CGGGT AACT CCC AGG AG AGT GTC A C AG AGC AGG AC AGC AAGG AC AGC ACCT AC AGCCT C AGC AGC ACCCT G ACGCT G AGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA (SEQ ID NO: 322).

[00737] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 123: [00738] CAGTCGGTGGAGGCGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACCTACTGGATGAGCTG GGT CCGCC AGGCTCC AGGGAAGGGGCT GGAAT GGAT CGGAGAC ATTT ATTTTAGT A ATGAAGAAACAAACTACGCGAGCTGGGCGAAAGGCCGATTTACCATCTCCAAAACC TCGACCACGGTGGATCTGAATGTCATCAGTCCGACAACCGAGGACACGGCCACCTA TTTCTGTGCCAGAGGTTCTCCTGATGTTGATATTGGTATAGATATGTGGGGCCCGGG CACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 323).

[00739] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 124:

[00740] CAGTCGGTGGAGGCGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCT G AC ACT C ACCTGC AC AGCCTCT GGATT CTCCCT C AGT ACCT ACT GGAT G AGCTG GGT CCGCC AGGCTCC AGGGAAGGGGCT GGAAT GGAT CGGAGAC ATTT ATTTT AGT A ATGAAGAAACAAACTACGCGAGCTGGGCGAAAGGCCGATTTACCATCTCCAAAACC 186 2016273912 14 Dec 2016

TCGACCACGGTGGATCTGAATGTCATCAGTCCGACAACCGAGGACACGGCCACCTA

TTTCTGTGCCAGAGGTTCTCCTGATGTTGATATTGGTATAGATATGTGGGGCCCGGG

CACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC

ACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG

ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC

GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG

GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC

AAGC CC AGC AAC AC C AAGGT GG AC AAGAGAGTT GAGCCC AAAT CTTGT GAC AAAAC

TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT

CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG

CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG

ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAG

CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA

GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA

TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC

CGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA

TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA

AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA

CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT

G AGGCTCT GC AC AACC ACTAC ACGC AG AAGAGCCT CT CCCTGT CT CCGGGT AAAT G A (SEQ ID NO: 324).

[00741] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 325; SEQ ID NO: 326; and SEQ ID NO: 327 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122.

[00742] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having 187 2016273912 14 Dec 2016 binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 328; SEQ ID NO: 329; and SEQ ID NO: 330 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124.

[00743] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 321 encoding the light chain variable sequence of SEQ ID NO: 121; the polynucleotide SEQ ID NO: 322 encoding the light chain sequence of SEQ ID NO: 122; the polynucleotide SEQ ID NO: 323 encoding the heavy chain variable sequence of SEQ ID NO: 123; the polynucleotide SEQ ID NO: 324 encoding the heavy chain sequence of SEQ ID NO: 124; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 325; SEQ ID NO: 326; and SEQ ID NO: 327) of the light chain variable sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 328; SEQ ID NO: 329; and SEQ ID NO: 330) of the heavy chain variable sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124.

[00744] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl3, the polynucleotides encoding the full length Abl3 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 322 encoding the light chain sequence of SEQ ID NO: 122 and the polynucleotide SEQ ID NO: 324 encoding the heavy chain sequence of SEQ ID NO: 124.

[00745] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such 188 2016273912 14 Dec 2016 as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl3 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl3 or Fab fragments thereof may be produced via expression of Abl3 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00746] Antibody Ab 14 [00747] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Abl4 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl4 polypeptides. The invention is further optionally directed to polynucleotides encoding antibody polypeptides having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 131: [00748] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA G AC AG AGT C ACC AT C ACTT GCC AGT CC AGTC AG AAT GTTT AT AAGAAC AACT ACTTA TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC ACT CT C ACC AT C AGC AGCCTGCAGCCTGAAGATGTTGC AACTTATT ACT GTGC AGGC GGTT AT ACC AGT AGT AGT GAT AATGCTTTCGGCGGAGGAACC AAGGT GG AAAT C AA ACGT (SEQ ID NO: 331).

[00749] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 132: 189 2016273912 14 Dec 2016 [00750] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA G AC AG AGTC ACC AT C ACTT GCC AGT CC AGTC AG AATGTTT AT AAGAAC AACT ACTT A TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC GGTT AT ACC AGT AGT AGT GAT AATGCTTTCGGCGGAGGAACC AAGGT GGAAAT C AA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGT AC AGT GG AAGGT GG AT A ACGC CCT CC AAT CGGGT AACT CCC AGG AG AGT GTC A CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 332).

[00751] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 133: [00752] G AGGT GC AGCTGGT GG AGT CT GGGGGAGGCTT GGT CC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTACCTACTGGATGAG CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGACATTTACTTTA GT AAT G A AG AAAC AAACT ACGC G AGC AGCGCGAAAGGCCGATT C ACC AT CT CC AG A G AC AATTCC AAGAAC AC CCT GT AT CTT C AAAT GAAC AGCCT GAG AGCT G AGG AC AC TGCTGTGTATTACTGTGCTAGAGGTTCTCCTGATGTTGATATTGGTATAGATATGTGG GGCCCAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 333).

[00753] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 134:

[00754] G AGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGT CC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTACCTACTGGATGAG CT GGGTCCGTC AGGCTCCAGGGAAGGGGCTGGAGT GGGTCGG AGACATTTACTTT A GT AAT G A AG AAAC AAACT ACGCGAGC AGCGCGAAAGGCCGATT C ACC AT CT CC AG A 190 2016273912 14 Dec 2016

G AC AATTCC AAG AAC AC CCT GT AT CTT C AAAT G AAC AGCCT G AG AGCT G AGG AC AC

TGCT GT GT ATT ACT GT GCT AG AGGTT CTCCT GAT GTT GAT ATT GGT AT AG AT AT GT GG

GGCCCAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTC

CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG

GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC

CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG

C AGCGT GGTG ACCGT GCCCTCC AGC AGCTT GGGC ACCC AG ACCT AC ATCT GC AACGT

GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTG

ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA

GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG

GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG

GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG

TACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG

AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA

GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC

CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA

GGCTTCT ATC CC AGCGAC ATCGCCGT GG AGT GGGAGAGC AAT GGGC AGCCGGAGAA

CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG

C AAGCT C ACCGT GGAC AAG AGC AGGT GGC AGC AGGGGAACGT CTT CT CAT GCT CCG

T GAT GC AT G AGGCT CTGC AC A ACC ACT AC ACGC AGAAGAGCCTCTCCCT GT CT CCGG GTAAATGA (SEQ ID NO: 334).

[00755] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 335; SEQ ID NO: 336; and SEQ ID NO: 337 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132.

[00756] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having 191 2016273912 14 Dec 2016 binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 338; SEQ ID NO: 339; and SEQ ID NO: 340 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134.

[00757] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 331 encoding the light chain variable sequence of SEQ ID NO: 131; the polynucleotide SEQ ID NO: 332 encoding the light chain sequence of SEQ ID NO: 132; the polynucleotide SEQ ID NO: 333 encoding the heavy chain variable sequence of SEQ ID NO: 133; the polynucleotide SEQ ID NO: 334 encoding the heavy chain sequence of SEQ ID NO: 134; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 335; SEQ ID NO: 336; and SEQ ID NO: 337) of the light chain variable sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 338; SEQ ID NO: 339; and SEQ ID NO: 340) of the heavy chain variable sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134.

[00758] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl4, the polynucleotides encoding the full length Abl4 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 332 encoding the light chain sequence of SEQ ID NO: 132 and the polynucleotide SEQ ID NO: 334 encoding the heavy chain sequence of SEQ ID NO: 134.

[00759] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such 192 2016273912 14 Dec 2016 as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl4 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl4 or Fab fragments thereof may be produced via expression of Abl4 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00760] Antibody Ab 15 [00761] The invention is further directed to the use of polynucleotides set forth below to produce antibody Abl5 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl5 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 141: [00762] GCAGCCGTGCTGACCCAGACACCATCGCCCGTGTCTGCAGCTGTGGGA G AC AC AGT C AC CAT C AAGT GCC AGT C C AGT C AG AGT GTTT AT AAG AAC AACT ACTT A TC CT GGT AT C AGC AG AAAC C AGGGC AGC CT C CC A AGCTCCT G ATCT ATG AT GC ATCC AAT CT GCC ATCTGGGGTCCC AT C ACGGTT C AGCGGC AGT GGATCTGGG AC AC AGTT C ACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC GATTATGATGATGATACTGATAATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAA ACGT (SEQ ID NO: 341).

[00763] In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 142: 193 2016273912 14 Dec 2016 [00764] GCAGCCGTGCTGACCCAGACACCATCGCCCGTGTCTGCAGCTGTGGGA G AC AC AGT C AC CAT C AAGT GCC AGTCC AGT C AGAGT GTTT AT AAGAAC AACTACTT A TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCC AATCTGCCATCTGGGGTCCCATCACGGTTCAGCGGCAGTGGATCTGGGACACAGTTC ACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC GATTATGATGATGATACTGATAATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA AT CT GGAACT GCCT CT GTT GT GT GCCTGCT GAATAACTT CT ATCCC AG AG AGGCC AA AGT AC AGT GGAAGGT GGAT A ACGCCCT CC AAT CGGGT AACT CCC AGG AG AGT GTC A C AGAGC AGGAC AGC AAGGAC AGC ACCT AC AGCCT C AGC AGC ACCCT G ACGCT G AGC AAAGC AGACT ACGAGAAAC AC AAAGT CT ACGCCTGCGAAGT C ACCC AT C AGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 342).

[00765] In another embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 143: [00766] C AGT C GGT GG AGG AGTCCGGGGGT CGCCT GGT C AC GCCT GGGAC ACC CCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCTATGCAATGATCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTTGGAGTGGTG GCACCTACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACC ACGGTGGATCTGCAAATCACCAGTCCGACAACCGAGGACGCGGCCACCTATTTCTGT GCCGCAGGTGGTGGTAGTATTTATGATGTTTGGGGCCCGGGCACCCTGGTCACCGTC TCGAGC (SEQ ID NO: 343).

[00767] In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 144:

[00768] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCTATGCAATGATCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTTGGAGTGGTG GCACCTACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACC 194 2016273912 14 Dec 2016

ACGGTGGATCTGCAAATCACCAGTCCGACAACCGAGGACGCGGCCACCTATTTCTGT

GCCGCAGGTGGTGGTAGTATTTATGATGTTTGGGGCCCGGGCACCCTGGTCACCGTC

TCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC

ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC

GGT GACGGT GTCGT GGAACTC AGGCGCCCTGACC AGCGGCGTGC ACACCTTCCCGG

CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA

GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC

AAGGT GG AC AAG AG AGTT GAGCCC AAAT CTT GT GAC AAAACT C AC AC ATGCCC ACC

GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC

CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGT

GAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC

ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTC

AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA

GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG

GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACC

AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT

GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAG

GT GGC AGC AGGGG AACGT CTTCT C ATGCTCCGT GAT GC AT G AGGCT CT GC AC AACC A CTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO: 344).

[00769] In a further embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 345; SEQ ID NO: 346; and SEQ ID NO: 347 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 141 or the light chain sequence of SEQ ID NO: 142.

[00770] In a further embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide 195 2016273912 14 Dec 2016 sequences of SEQ ID NO: 348; SEQ ID NO: 349; and SEQ ID NO: 350 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 143 or the heavy chain sequence of SEQ ID NO: 144.

[00771] The invention also contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 341 encoding the light chain variable sequence of SEQ ID NO: 141; the polynucleotide SEQ ID NO: 342 encoding the light chain sequence of SEQ ID NO: 142; the polynucleotide SEQ ID NO: 343 encoding the heavy chain variable sequence of SEQ ID NO: 143; the polynucleotide SEQ ID NO: 344 encoding the heavy chain sequence of SEQ ID NO: 144; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 345; SEQ ID NO: 346; and SEQ ID NO: 347) of the light chain variable sequence of SEQ ID NO: 141 or the light chain sequence of SEQ ID NO: 142; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 348; SEQ ID NO: 349; and SEQ ID NO: 350) of the heavy chain variable sequence of SEQ ID NO: 143 or the heavy chain sequence of SEQ ID NO: 144.

[00772] In a preferred embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl5, the polynucleotides encoding the full length Abl5 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 342 encoding the light chain sequence of SEQ ID NO: 142 and the polynucleotide SEQ ID NO: 344 encoding the heavy chain sequence of SEQ ID NO: 144.

[00773] Another embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast 196 2016273912 14 Dec 2016

Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl5 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl5 or Fab fragments thereof may be produced via expression of Abl5 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00774] Antibody Abl6 [00775] The invention is further directed to the use of polynucleotides set forth below to produce antibody Abl6 polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF, which inhibit the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl6 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 151: [00776] GCCCTGGTGATGACCCAGACTCCATCCTCCACGTCTGAACCAGTGGGA GGC AC AGT C ACC ATC AATT GCC AGGCT AGT C AGAAT ATT GGT AACG ACCTAT CCT GG T ATC AGC AG AAACC AGGGC AGCCT CCCG AGCT CCT AAT CT ATT CT AC AT CC AAACT G GCAACTGGGGTCCCAAAGCGGTTCAGTGGCAGCAGATCTGGGACACAGTTCACTCT CACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCTAGGTGTTTA TAGTTATATTAGTGATGATGGTAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA ACGT (SEQ ID NO: 351).

[00777] In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 152: 197 2016273912 14 Dec 2016 [00778] GCCCTGGTGATGACCCAGACTCCATCCTCCACGTCTGAACCAGTGGGA GGC AC AGT C ACC ATC AATTGCC AGGCT AGT C AGAAT ATT GGT AACG ACCTAT CCT GG T ATC AGC AGAAACC AGGGC AGCCT CCCGAGCT CCT AAT CT ATT CT AC AT CC AAACT G GC AACT GGGGT CCC AAAGCGGTT C AGT GGC AGC AGATCT GGGAC AC AGTT C ACTCT CACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCTAGGTGTTTA TAGTTATATTAGTGATGATGGTAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGT AC AGT GG AAGGT GGATAACGCCCT CC AAT CGGGT AACT CCC AGG AG AGT GTC A C AG AGC AGG AC AGC AAGG AC AGC ACCT AC AGCCT C AGC AGC ACCCT G ACGCT G AGC AAAGC AG ACT ACG AG A AAC AC AAAGT CT ACGCCT GCG AAGT C ACCC AT C AGGGCCT G AGCTC GCCC GT C AC AA AG AGCTT C AAC AGGGG AG AGT GTT AG (SEQ ID NO: 352).

[00779] In another embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 153: [00780] CAGTCGGTGGAGGAGTTCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAATAACTATGCAATGACCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGGATCATTGGTAGTATTG GTACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCTTCATCTCCAAAACCTCGA CCACTGTGGATCTGAAAATCATTAGTCCGACAACCGAGGACACGGCCACCTATTTCT GT GCC AG AG AT GCTGGCGTTACT GTT GAT GGTT AT GGCT ACT ACTTT AAC AT CTGGG GCCCAGGCACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 353).

[00781] In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 154:

[00782] CAGTCGGTGGAGGAGTTCGGGGGTCGCCTGGTCACGCCTGGGACACC CCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAATAACTATGCAATGACCTG GGT CCGCC AGGCT CC AGGGAAGGGGCT GGAGT GG ATCGGG AT C ATT GGT AGTATTG GTACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCTTCATCTCCAAAACCTCGA 198 2016273912 14 Dec 2016

CCACTGTGGATCTGAAAATCATTAGTCCGACAACCGAGGACACGGCCACCTATTTCT

GT GCC AGAG ATGCTGGCGTTACT GTT GAT GGTT AT GGCT ACT ACTTT AAC AT CT GGG

GCCCAGGCACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCC

CCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG

TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC

AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC

AGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG

AATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA

C AAAACT C AC AC AT GCCC ACCGT GCCC AGC ACCT G AACT CCT GGGGGGACCGT C AG

TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG

T C AC AT GCGT GGT GGTGG ACGT G AGCC ACG AAG ACCCT G AGGT C AAGTT C AACTGG

T ACGT GGACGGCGT GG AGGT GC AT A ATGCC AAG AC AAAGCCGCGGGAGGAGC AGT

ACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA

ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG

AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC

CCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG

GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC

AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC

AAGCT C ACCGT GG AC AAG AGC AGGT GGC AGC AGGGGAACGT CTT CT CAT GCT CCGT

GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAATGA (SEQ ID NO: 354).

[00783] In a further embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 355; SEQ ID NO: 356; and SEQ ID NO: 357 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 151 or the light chain sequence of SEQ ID NO: 152.

[00784] In a further embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding 199 2016273912 14 Dec 2016 specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 358; SEQ ID NO: 359; and SEQ ID NO: 360 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 153 or the heavy chain sequence of SEQ ID NO: 154.

[00785] The invention also contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 351 encoding the light chain variable sequence of SEQ ID NO: 151; the polynucleotide SEQ ID NO: 352 encoding the light chain sequence of SEQ ID NO: 152; the polynucleotide SEQ ID NO: 353 encoding the heavy chain variable sequence of SEQ ID NO: 153; the polynucleotide SEQ ID NO: 354 encoding the heavy chain sequence of SEQ ID NO: 154; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 355; SEQ ID NO: 356; and SEQ ID NO: 357) of the light chain variable sequence of SEQ ID NO: 151 or the light chain sequence of SEQ ID NO: 152; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 358; SEQ ID NO: 359; and SEQ ID NO: 360) of the heavy chain variable sequence of SEQ ID NO: 153 or the heavy chain sequence of SEQ ID NO: 154.

[00786] In a preferred embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl6, the polynucleotides encoding the full length Abl6 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 352 encoding the light chain sequence of SEQ ID NO: 152 and the polynucleotide SEQ ID NO: 354 encoding the heavy chain sequence of SEQ ID NO: 154.

[00787] Another embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, 200 2016273912 14 Dec 2016 HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl6 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl6 or Fab fragments thereof may be produced via expression of Abl6 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00788] Antibody Ab 17 [00789] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Abl7 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA without appreciably inhibiting the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl7 polypeptides. The invention is further optionally directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 161: [00790] GCCATCGAAATGACCCAGACTCCATTCTCCGTGTCTGCAGCTGTGGGA GGC AC AGT C ACC AT C AAGT GC C AGGCC AGT C AG AC C ATT AGC AACT ACTT AGC CTG GT ATC AGC AGAAACC AGGGC AGCCTCCC AAGCTCCTGAT CT ATGGT GC ATCC AATCT GG AAT CT GGGGT C CC AT C GCGGTT C AAAGGC AGT GG ATCTGGG AC AC AGTT C ACT CT CACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGGTTA TACT AT C AGT AAT GTT GAT AAC AAT GTTTT C GGCGG AGGG ACCG AGGTGGT GGT C AA ACGT (SEQ ID NO: 361). 201 2016273912 14 Dec 2016 [00791] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 162: [00792] GCCATCGAAATGACCCAGACTCCATTCTCCGTGTCTGCAGCTGTGGGA GGCACAGTCACCATCAAGTGCCAGGCCAGTCAGACCATTAGCAACTACTTAGCCTG GTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCAATCT GGAATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCT CACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGGTTA TACTATCAGTAATGTTGATAACAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA AT CT GGAACT GCCT CT GTT GT GT GCCTGCT G AAT AACTT CT ATCCC AGAGAGGCC AA AGT AC AGT GGAAGGT GG AT A ACGCCCT CC AAT CGGGT AACT CCC AGGAGAGT GTC A CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGC AGACT ACG AG A AAC AC AAAGT CT ACGCCT GCG AAGT C ACCC AT C AGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 362).

[00793] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 163: [00794] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGGGATC CCTGACACTCACCTGCGCAGCCTCTGGATTCTCCCTCACTGGCTACAACTTGGTCTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATTCATTAGTTATGGTG ATACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG ACC ACGGT GACT CT GACGATC ACCG ATCTGC AACCTT C AGAC ACGGGC ACCT ATTTC TGTGCCAGAGAGACTGCTAATACTTATGATTATGGCATCTGGGGCCCAGGCACCCTC GTCACCGTCTCGAGC (SEQ ID NO: 363).

[00795] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 164: 202 2016273912 14 Dec 2016 [00796] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGGGATC CCTGACACTCACCTGCGCAGCCTCTGGATTCTCCCTCACTGGCTACAACTTGGTCTG GGTCCGCC AGGCTCC AGGGAAGGGGCT GGAGT GGATCGGATTC ATT AGTT ATGGT G ATACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCG ACCACGGTGACTCTGACGATCACCGATCTGCAACCTTCAGACACGGGCACCTATTTC TGTGCCAGAGAGACTGCTAATACTTATGATTATGGCATCTGGGGCCCAGGCACCCTC GTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTT CCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACA CCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA GCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACA TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGT GG ACGT G AGC C ACG AAG ACC CT G AGGT C AAGTT C AACT GGT ACGT GGACGGCG TGG AGGT GC AT AAT GC C AAG AC AAAGCC GCGGG AGGAGC AGT ACGCC AGC ACGT A CCGTGTGGTC AGCGTCCTC ACCGTCCTGC ACC AGG ACT GGCT G AAT GGC AAGG AGT ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT GG AC A AG AGC AGGT GGC AGC AGGGG AAC GTCTT CT CAT GCTCCGT GAT GC AT G AGG CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO: 364).

[00797] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 365; SEQ ID NO: 366; and SEQ ID NO: 367 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or 203 2016273912 14 Dec 2016 hypervariable regions) of the light chain variable sequence of SEQ ID NO: 161 or the light chain sequence of SEQ ID NO: 162.

[00798] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 368; SEQ ID NO: 369; and SEQ ID NO: 370 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 163 or the heavy chain sequence of SEQ ID NO: 164.

[00799] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 361 encoding the light chain variable sequence of SEQ ID NO: 161; the polynucleotide SEQ ID NO: 362 encoding the light chain sequence of SEQ ID NO: 162; the polynucleotide SEQ ID NO: 363 encoding the heavy chain variable sequence of SEQ ID NO: 163; the polynucleotide SEQ ID NO: 364 encoding the heavy chain sequence of SEQ ID NO: 164; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 365; SEQ ID NO: 366; and SEQ ID NO: 367) of the light chain variable sequence of SEQ ID NO: 161 or the light chain sequence of SEQ ID NO: 162; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 368; SEQ ID NO: 369; and SEQ ID NO: 370) of the heavy chain variable sequence of SEQ ID NO: 163 or the heavy chain sequence of SEQ ID NO: 164.

[00800] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl7, the polynucleotides encoding the full length Abl7 antibody comprise, or alternatively consist of, the 204 2016273912 14 Dec 2016 polynucleotide SEQ ID NO: 362 encoding the light chain sequence of SEQ ID NO: 162 and the polynucleotide SEQ ID NO: 364 encoding the heavy chain sequence of SEQ ID NO: 164.

[00801] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris.

In one optional embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl7 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl7 or Fab fragments thereof may be produced via expression of Abl7 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00802] Antibody Abl 8 [00803] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Abl8 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Abl8 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 171:

[00804] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGTCAGGCTAGTCAGACCATTAGCAACTACTTAGCCTGG TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATCTG G AAT CT GGAGT CCC AT C AAGGTTC AGCGGC AGT GG AT CT GGAAC AGAATT C ACTCTC ACC ATC AGC AGCCT GC AGCCT GAT G ATTTT GC AACTT ACT ACT GTC AAC AGGGTT AT 205 2016273912 14 Dec 2016 ACTATCAGTAATGTTGATAACAATGTTTTCGGCGGAGGAACCAAGGTGGAAATCAA ACGT (SEQ ID NO: 371).

[00805] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 172: [00806] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA G AC AGAGTC ACC AT C ACTT GT C AGGCT AGT C AGACC ATTAGC AACT ACTT AGCCTGG TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATCTG GAATCTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGAACAGAATTCACTCTC ACC ATC AGC AGCCT GC AGCCT GAT G ATTTT GC AACTT ACT ACT GTC AAC AGGGTT AT ACTATCAGTAATGTTGATAACAATGTTTTCGGCGGAGGAACCAAGGTGGAAATCAA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA AT CT GGAACT GCCTCT GTT GT GT GCCTGCT G AAT AACTT CT ATCCC AGAGAGGCC AA AGT AC AGT GG AAGGT GG AT A ACGCCCT CC AAT CGGGT AACT CCC AGGAGAGT GTC A CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 372).

[00807] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 173: [00808] GAGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGTCC AGCCTGGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTGGCTACAACTTGGT CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGATTCATTAGTTATG GTGATACCACATACTACGCTAGCTCTGCTAAAGGCCGATTCACCATCTCCAGAGACA ATTC C AAG AAC ACC CT GT AT CTT C AA AT G AAC AGC CT G AG AGCT G AGG AC ACTGCTG T GT ATT ACT GT GCT AG AG AG ACT GCT AAT ACTT AT GATT AT GGC AT CT GGGGCC AAG GGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 373). 206 2016273912 14 Dec 2016 [00809] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 174: [00810] GAGGT GC AGCTGGT GGAGTCT GGGGGAGGCTT GGTCC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTGGCTACAACTTGGT CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGATTCATTAGTTATG GTGATACCACATACTACGCTAGCTCTGCTAAAGGCCGATTCACCATCTCCAGAGACA ATT C C AAG AAC ACC CT GT AT CTTC AA AT G AAC AGC CT G AG AGCT G AGGAC ACTGCT G T GT ATT ACT GT GCT AG AG AG ACT GCT AAT ACTT AT GATT AT GGC AT CT GGGGCC AAG GGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA C AAGC CC AGC AAC ACC AAGGT GGAC AAG AG AGTT G AGCCC AAAT CTT GT G AC AAAA CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCA GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATC CCGGG AGGAGAT GACC AAG AACC AGGT C AGCCT GACCT GCCT GGT C AAAGGCTT CT ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA T GAGGCT CT GC AC AACC ACT AC ACGC AG AAG AGCCT CTCCCT GT CT CCGGGT AAAT G A (SEQ ID NO: 374).

[00811] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having 207 2016273912 14 Dec 2016 binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 375; SEQ ID NO: 376; and SEQ ID NO: 377 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 171 or the light chain sequence of SEQ ID NO: 172.

[00812] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 378; SEQ ID NO: 379; and SEQ ID NO: 380 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 173 or the heavy chain sequence of SEQ ID NO: 174.

[00813] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 371 encoding the light chain variable sequence of SEQ ID NO: 171; the polynucleotide SEQ ID NO: 372 encoding the light chain sequence of SEQ ID NO: 172; the polynucleotide SEQ ID NO: 373 encoding the heavy chain variable sequence of SEQ ID NO: 173; the polynucleotide SEQ ID NO: 374 encoding the heavy chain sequence of SEQ ID NO: 174; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 375; SEQ ID NO: 376; and SEQ ID NO: 377) of the light chain variable sequence of SEQ ID NO: 171 or the light chain sequence of SEQ ID NO: 172; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 378; SEQ ID NO: 379; and SEQ ID NO: 380) of the heavy chain variable sequence of SEQ ID NO: 173 or the heavy chain sequence of SEQ ID NO: 174. 208 2016273912 14 Dec 2016 [00814] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments for treatment or prevention of pain and pain associated conditions having binding specificity for NGF. With respect to antibody Abl8, the polynucleotides encoding the full length Abl8 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 372 encoding the light chain sequence of SEQ ID NO: 172 and the polynucleotide SEQ ID NO: 374 encoding the heavy chain sequence of SEQ ID NO: 174.

[00815] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl8 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl8 or Fab fragments thereof may be produced via expression of Abl8 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00816] Antibody Abl 9 [00817] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Abl9 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75. in methods of treating pain in an individual comprising administering to said individual antibody Abl9 polypeptides. The invention is further optionally directed to polynucleotides encoding antibody polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 181: 209 2016273912 14 Dec 2016 [00818] GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGA GGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAATGTTTATAAGAACAACTATTTA TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCTTCC ACTCT GGC AT CT GGGGTCCC ATCGCGGTT C AAAGGC AGT GG ATCTGGG AC AG ATTTC ACT CT C AC CAT C AGCG AC GT GC AGT GT G ACGCT GCT GCC ACTT ACT ACT GT GC AGGC GGTT AT AGT AGT AGT AGT GAT AAT GCTTTC GGCGG AGGG ACCG AGGT GGT GGT C AA ACGT (SEQ ID NO: 381).

[00819] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 182: [00820] GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGA GGC AC AGT C AGC AT C AGTT GCC AGTCC AGT C AGAAT GTTT AT AAG AAC AACT ATTT A TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCTTCC ACT CT GGC AT CT GGGGTCCC AT CGCGGTT C AAAGGC AGT GGATCT GGG AC AG ATTT C ACT CT C AC CAT C AGCG AC GT GC AGT GT G ACGCT GCT GCC ACTT ACT ACT GT GC AGGC GGTT AT AGT AGT AGT AGT GAT AAT GCTTTCGGCGG AGGG ACCG AGGTGGT GGT C AA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA AT CT GGAACT GCCTCT GTT GT GTGCCTGCT G AATAACTTCT ATCCC AGAGAGGCC AA AGT AC AGT GG AAGGT GG AT A ACGCCCT CC AAT CGGGT AACT CCC AGGAGAGT GTC A CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 382).

[00821] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 183:

[00822] CAGTCGGTGGAGGCGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCC CTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACCTACTGGATGTCCTGG GTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGACATTTATTTTAGTAA TGAGGAAACAAACTACGCGACCTGGGCGAAAGGCCGATTTACCATCTCCAAAACCT 210 2016273912 14 Dec 2016 CGACCACGGTGGATCTGAATGTCATCAGTCCGACAACCGAGGACACGGCCACCTAT TTCT GT GC AAG AGGTT CTC CT GAT GTT GAG ATT GCT AT AG AT AT GT GGGGCC AGGGC ACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 383).

[00823] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 184:

[00824] CAGTCGGTGGAGGCGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCC CTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTACCTACTGGATGTCCTGG GTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGACATTTATTTTAGTAA TGAGGAAACAAACTACGCGACCTGGGCGAAAGGCCGATTTACCATCTCCAAAACCT CGACCACGGTGGATCTGAATGTCATCAGTCCGACAACCGAGGACACGGCCACCTAT TTCT GT GC AAG AGGTT CTC CT GAT GTT GAG ATT GCT AT AG AT AT GT GGGGCC AGGGC ACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACA AGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT CTCC AAAGCC AAAGGGC AGCCCCG AG AACC AC AGGT GT AC ACCCT GCCCCC ATCCC GGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACC ACGCCT CCCGT GCT GG ACT CCG ACGGCT CCTT CTTCCT CT AC AGC AAGCT C A CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT 211 2016273912 14 Dec 2016 GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATG A (SEQ ID NO: 384).

[00825] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 385; SEQ ID NO: 386; and SEQ ID NO: 387 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 181 or the light chain sequence of SEQ ID NO: 182.

[00826] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 388; SEQ ID NO: 389; and SEQ ID NO: 390 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 183 or the heavy chain sequence of SEQ ID NO: 184.

[00827] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 381 encoding the light chain variable sequence of SEQ ID NO: 181; the polynucleotide SEQ ID NO: 382 encoding the light chain sequence of SEQ ID NO: 182; the polynucleotide SEQ ID NO: 383 encoding the heavy chain variable sequence of SEQ ID NO: 183; the polynucleotide SEQ ID NO: 384 encoding the heavy chain sequence of SEQ ID NO: 184; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 385; SEQ ID NO: 386; and SEQ ID NO: 387) of the light chain variable sequence of SEQ ID NO: 181 or the light chain sequence of SEQ ID NO: 182; and 212 2016273912 14 Dec 2016 polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 388; SEQ ID NO: 389; and SEQ ID NO: 390) of the heavy chain variable sequence of SEQ ID NO: 183 or the heavy chain sequence of SEQ ID NO: 184.

[00828] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Abl9, the polynucleotides encoding the full length Abl9 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 382 encoding the light chain sequence of SEQ ID NO: 182 and the polynucleotide SEQ ID NO: 384 encoding the heavy chain sequence of SEQ ID NO: 184.

[00829] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one optional embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl9 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Abl9 or Fab fragments thereof may be produced via expression of Abl9 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00830] Antibody Ab20 [00831] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Ab20 polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF, which inhibits the association of NGF with TrkA and the association of NGF with p75, in methods of treating pain in an individual comprising administering to said individual antibody Ab20 polypeptides. The invention is further directed to polynucleotides encoding antibody polypeptides having binding specificity to NGF. In one optional embodiment of the invention, polynucleotides of the invention comprise, 213 2016273912 14 Dec 2016 or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 191: [00832] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA GAC AGAGTC ACC AT C ACTT GCC AGT CC AGTC AG AATGTTT AT AAGAAC AACT ACTT A TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC GGTTATACCAGTAGTAGTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAA ACGT (SEQ ID NO: 391).

[00833] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 192: [00834] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA GAC AG AGT C ACC AT C ACTT GCC AGT CC AGTC AG AAT GTTT AT AAGAAC AACT ACTT A TCCTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATAAGGCATCC ACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGACAGATTTC ACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGC GGTT AT ACC AGT AGT AGT GAT AATGCTTTCGGCGGAGGAACC AAGGT GGAAAT C AA ACGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGT AC AGT GGAAGGT GGATAACGCCCT CC AAT CGGGT AACT CCC AGGAGAGT GTC A CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGC AG ACT ACGAGAAAC AC AAAGT CT ACGCCT GCG AAGT C ACCC AT C AGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 392).

[00835] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 193: 214 2016273912 14 Dec 2016 [00836] GAGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGT CC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTACCTACTGGATGAG CT GGGT CCGTC AGGCT CC AGGGAAGGGGCT GGAGT GGGT CGG AGAC ATTT ACTTT A GTAATGAAGAAACAAACTACGCGACCAGCGCGAAAGGCCGATTCACCATCTCCAGA GAC AATT CC AAGAAC ACCCT GTAT CTT C AAAT GAAC AGCCT G AGAGCT GAGGAC AC TGCT GTGT ATT ACT GTGCTAGAGGTT CTCCT GAT GTTGAG ATT GCTAT AGAT ATGTGG GGCCAAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 393).

[00837] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 194:

[00838] GAGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGT CC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTACCTACTGGATGAG CT GGGTCCGT C AGGCT CC AGGGAAGGGGCT GGAGT GGGT CGG AG AC ATTT ACTTT A GTAATGAAGAAACAAACTACGCGACCAGCGCGAAAGGCCGATTCACCATCTCCAGA G AC AATTCC AAGAAC AC CCT GT AT CTT C AAAT GAAC AGCCT G AG AGCT GAGGAC AC TGCTGTGT ATT ACTGTGCTAGAGGTTCTCCTGATGTTGAGATTGCTATAGAT ATGTGG GGCCAAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT G AAT C AC AAGCC C AGC AAC AC C AAGGT GGAC AAGAGAGTTG AGCCC AAATCTT GT G ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG TACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA 215 2016273912 14 Dec 2016 GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG C AAGCT C ACCGT GGAC AAGAGC AGGT GGC AGC AGGGG AACGT CTT CT CAT GCTCCG TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGA (SEQ ID NO: 394).

[00839] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 395; SEQ ID NO: 396; and SEQ ID NO: 397 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 191 or the light chain sequence of SEQ ID NO: 192.

[00840] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 398; SEQ ID NO: 399; and SEQ ID NO: 400 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 193 or the heavy chain sequence of SEQ ID NO: 194.

[00841] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 391 encoding the light chain variable sequence of SEQ ID NO: 191; the polynucleotide SEQ ID NO: 392 encoding the light chain sequence of SEQ ID NO: 192; the polynucleotide SEQ ID NO: 393 encoding the heavy chain variable sequence of SEQ ID NO: 193; the polynucleotide SEQ ID NO: 394 encoding the heavy chain 216 2016273912 14 Dec 2016 sequence of SEQ ID NO: 194; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 395; SEQ ID NO: 396; and SEQ ID NO: 397) of the light chain variable sequence of SEQ ID NO: 191 or the light chain sequence of SEQ ID NO: 192; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 398; SEQ ID NO: 399; and SEQ ID NO: 400) of the heavy chain variable sequence of SEQ ID NO: 193 or the heavy chain sequence of SEQ ID NO: 194.

[00842] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab20, the polynucleotides encoding the full length Ab20 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 392 encoding the light chain sequence of SEQ ID NO: 192 and the polynucleotide SEQ ID NO: 394 encoding the heavy chain sequence of SEQ ID NO: 194.

[00843] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab20 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Ab20 or Fab fragments thereof may be produced via expression of Ab20 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00844] Antibody Ab21 [00845] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody Ab21 polypeptides having binding specificity to NGF, which inhibit the association of NGF with TrkA and the association of NGF with p75 in methods of treating pain in an individual comprising administering to said individual antibody Ab21 polypeptides. The invention is further optionally directed to polynucleotides encoding antibody polypeptides 217 2016273912 14 Dec 2016 for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 51: [00846] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG GAAT CT GGAGT CCC AT C AAGGTTC AGCGGC AGT GGAT CT GGG AC AG AGT AC ACT CT CACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTACTACTGCCAACAGGGTTT TACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC GT (SEQ ID NO: 251).

[00847] In one embodiment of the invention, polynucleotides of the invention optionally comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 401: [00848] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG GAAT CT GG AGTCC C AT C AAGGTT C AGCGGC AGT GGAT CT GGG AC AG AGT AC ACT CT C ACC ATC AGC AGCCTGC AGCCT GAT GATTTTGC AACTT ACT ACT GCC AAC AGGGTTT TACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 403).

[00849] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 53: 218 2016273912 14 Dec 2016 [00850] GAGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGT CC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTATTACGTCTTTAACATCT GGGGCCCAGGGACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 253).

[00851] In one optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 402:

[00852] GAGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGT CC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC AATT C C AAG AAC AC CCT GT AT CTTC AAAT GAAC AGCCT G AG AGCT G AGG AC ACTGCT GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTACTACGTCTTTAACATCT GGGGCCCAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCT TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA CGT GAAT CAC AAGCCCAGCAAC ACC AAGGTGGACGCGAGAGTTGAGCCC AAATCTT GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC AGT ACGCC AGC ACGT ACCGT GT GGTC AGCGT CCTC ACCGT CCT GC ACC AGGACT GGC T GAATGGC AAGGAGT AC AAGT GC AAGGT CT CC AAC AAAGCCCT CCC AGCCCCC AT C GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA 219 2016273912 14 Dec 2016 AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA (SEQ ID NO: 404).

[00853] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 401.

[00854] In a further optional embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one or more of the polynucleotide sequences of SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 402.

[00855] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 251 encoding the light chain variable sequence of SEQ ID NO: 51; the polynucleotide SEQ ID NO: 403 encoding the light chain sequence of SEQ ID NO: 401; the polynucleotide SEQ ID NO: 253 encoding the heavy chain variable sequence of SEQ ID NO: 53; the polynucleotide SEQ ID NO: 404 encoding the heavy chain sequence of 220 2016273912 14 Dec 2016 SEQ ID NO: 402; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257) of the light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 401; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260) of the heavy chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 402.

[00856] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody Ab21, the polynucleotides encoding the full length Ab21 antibody comprise, or alternatively consist of, the polynucleotide SEQ ID NO: 403 encoding the light chain sequence of SEQ ID NO: 401 and the polynucleotide SEQ ID NO: 404 encoding the heavy chain sequence of SEQ ID NO: 402.

[00857] Another optional embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one optional embodiment of the invention described herein (infra), Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab21 following expression of the full-length polynucleotides in a suitable host. In another embodiment of the invention, anti-NGF antibodies such as Ab21 or Fab fragments thereof may be produced via expression of Ab21 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00858] Antibody fragment Fab2 [00859] The invention is further optionally directed to the use of polynucleotides set forth below to produce antibody fragment Fab2 polypeptides that inhibit the association of NGF with TrkA and p75 for treatment or prevention of pain and pain associated conditions having binding specificity to NGF in methods of treating pain in an individual comprising administering to said individual antibody Abl polypeptides . The invention is further directed to polynucleotides 221 2016273912 14 Dec 2016 encoding antibody fragment polypeptides for treatment or prevention of pain and pain associated conditions having binding specificity to NGF. In one embodiment of the invention, Fab polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the light chain polypeptide sequence of SEQ ID NO: 407: [00860] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTTACAGCAATCTTGCCTGG TATCAGCAGAAACCAGGAAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTCTG G AAT CT GGAGT CCC AT C AAGGTTC AGCGGC AGT GG AT CT GGG AC AGAGT AC ACT CT C AC CAT C AGC AGCCTGC AGCCT GAT G ATTTTGC AACTT ACT ACT GCC AAC AGGGTTT TACTGTTAGTGATATTGATAATGCTTTCGGCGGAGGAACCAAGGTGGAAATCAAAC GTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG T AC AGT GGAAGGT GGAT AACGCCCT CC AAT CGGGT AACT CCC AGGAGAGTGT C AC A GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA AGC AG ACT ACGAGAAAC AC AAAGTCT ACGCCT GCG AAGT C ACCC AT C AGGGCCT G A GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 409).

[00861] In another optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, the following polynucleotide sequence encoding the heavy chain polypeptide sequence of SEQ ID NO: 408:

[00862] GAGGT GC AGCTGGT GGAGT CT GGGGGAGGCTT GGTCC AGCCT GGGGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAACTATGCAGTGGG CTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAATCATTGGTCGTA ATGGTAACACATGGTACGCGAGCTCTGCAAGAGGCCGATTCACCATCTCCAGAGAC AATTCCAAGAACACCCTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGACACTGCT GTGTATTACTGTGCTAGAGGATATGGCCGTAGTGTTGCTTACTACGTCTTTAACATCT GGGGCCCAGGGACCCTCGTCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCT TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC 222 2016273912 14 Dec 2016 AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA CGTGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCTT GT G AC AAAACT C ACT AG (SEQ ID NO: 410).

[00863] In a further optional embodiment of the invention, polynucleotides encoding Fab antibody fragments having binding specificity to NGF comprise one or more of the polynucleotide sequences of SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 409.

[00864] In a further optional embodiment of the invention, polynucleotides encoding Fab antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise one or more of the polynucleotide sequences of SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 410.

[00865] The invention also optionally contemplates polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments for treatment or prevention of pain and pain associated conditions described herein. In one embodiment of the invention, polynucleotides encoding antibody fragments for treatment or prevention of pain and pain associated conditions having binding specificity to NGF comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 251 encoding the light chain variable sequence of SEQ ID NO: 51; the polynucleotide SEQ ID NO: 409 encoding the light chain sequence of SEQ ID NO: 407; the polynucleotide SEQ ID NO: 253 encoding the heavy chain variable sequence of SEQ ID NO: 53; the polynucleotide SEQ ID NO: 410 encoding the heavy chain sequence of SEQ ID NO: 408; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID NO: 257) of the light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 407; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 223 2016273912 14 Dec 2016 260) of the heavy chain variable sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 408.

[00866] In a preferred optional embodiment of the invention, polynucleotides of the invention comprise, or alternatively consist of, polynucleotides encoding Fab (fragment antigen binding) fragments having binding specificity for NGF. With respect to antibody fragment Fab2, the polynucleotides encoding the Fab fragment include the polynucleotide SEQ ID NO: 409 encoding the light chain sequence of SEQ ID NO: 407 and the polynucleotide SEQ ID NO: 410 encoding the heavy chain sequence of SEQ ID NO: 408.

[00867] Another embodiment of the invention contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, plant or microbial systems such as yeast cells such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the invention described herein (infra), Fab fragments may be produced via expression of Fab2 polynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, plant or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[00868] In one embodiment, the invention is optionally directed to an isolated polynucleotide comprising a polynucleotide encoding an anti-NGF VH antibody amino acid sequence selected from SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, or 402, or encoding a variant thereof wherein at least one framework residue (FR residue) has been substituted with an amino acid present at the corresponding position in a rabbit anti-NGF antibody Vh polypeptide or a conservative amino acid substitution.

[00869] In another optional embodiment, the invention is directed to an isolated polynucleotide comprising the polynucleotide sequence encoding an anti-NGF Vl antibody amino acid sequence ofl, 11,21,31,41,51,61,71,81,91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, or 401, or encoding a variant thereof wherein at least one framework residue (FR 224 2016273912 14 Dec 2016 residue) has been substituted with an amino acid present at the corresponding position in a rabbit anti-NGF antibody VL polypeptide or a conservative amino acid substitution.

[00870] In yet another optional embodiment, the invention is directed to one or more heterologous polynucleotides comprising a sequence encoding the polypeptides contained in SEQ ID NO:l and SEQ ID NO:3; SEQ ID NO:l 1 and SEQ ID NO: 13; SEQ ID NO:21 and SEQ ID NO:23; SEQ ID NO:31 and SEQ ID NO:33; SEQ ID NO:411 and SEQ ID NO:43; SEQ ID NO:51 and SEQ ID NO:53, SEQ ID NO:61 and SEQ ID NO:63; SEQ ID NO:71 and SEQ ID NO:73; SEQ ID NO:81 and SEQ ID NO:83; SEQ ID NO:91 and SEQ ID NO:93; SEQ ID NO:101 and SEQ IDNO:103; SEQ ID NO:lll and SEQ IDNO:113; SEQ IDNO:121 and SEQ ID NO: 123; SEQ ID NO: 131 and SEQ ID NO: 133; SEQ ID NO: 141 and SEQ ID NO: 143; SEQ ID NO:151 and SEQ IDNO:153;SEQ IDNO:161 and SEQ ID NO: 163; SEQ ID NO: 171 and SEQ ID NO:173; SEQ ID NO:181 and SEQ ID NO:183; SEQ ID NO:191 and SEQ ID NO:193; or SEQ ID NO:401 and SEQ ID NO:403.

[00871] In another embodiment, the invention is optionally directed to an isolated polynucleotide that expresses a polypeptide containing at least one CDR polypeptide derived from an anti-NGF antibody wherein said expressed polypeptide alone specifically binds NGF or specifically binds NGF when expressed in association with another polynucleotide sequence that expresses a polypeptide containing at least one CDR polypeptide derived from an anti-NGF antibody for treatment or prevention of pain and pain associated conditions wherein said at least one CDR is selected from those contained in the Vl or VH polypeptides of SEQ ID NO: 1,3, 11, 13,21,23,31,33,41,43,51,53,61,63,71,73,81,83,91,93, 101, 103, 111, 113, 121, 123, 131, 133, 141, 143, 151, 153, 161, 163, 171, 173, 181, 183, 191, 193,401 or SEQ IDNO:403.

[00872] Host cells and vectors comprising said polynucleotides are also contemplated.

[00873] The invention further optionally contemplates vectors comprising the polynucleotide sequences encoding the variable heavy and light chain polypeptide sequences, as well as the individual complementarity-determining regions (CDRs, or hypervariable regions), as set forth herein, as well as host cells comprising said vector sequences. In one embodiment of the invention, the host cell is a yeast cell. In another embodiment of the invention, the yeast host cell belongs to the genus Pichia. 225 2016273912 14 Dec 2016

EXAMPLES

[00874] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

[00875] EXAMPLE 1 [00876] This example describes culture methods that improved the purity of recombinant antibodies produced from cultured P. pastoris cells. When a bolus of ethanol was added during culturing, the resulting antibodies exhibited a great decrease in the concentration of an undesired product-associated variant.

[00877] Methods [00878] To generate the inoculum, diploid P. pastoris was grown using a medium composed of the following nutrients (percentages are given as w/v): yeast extract 3%, anhydrous dextrose 2%, YNB 1.34%, Biotin 0.004% and 100 mM potassium phosphate (pH 6.0). The inoculum medium for runs L355, L357, L358, L359 and L360 was composed of the following nutrients (percentages are given as w/v): yeast extract 3%, glycerol 2%, YNB 1.34%, Biotin 0.004%, 200 mM potassium phosphate (pH 6.0) The inoculum was grown for approximately 24 hours to 29 hours in a shaking incubator at 30°C and 300 rpm. A 10% inoculum was then added to Labfors 2.5L working volume vessels containing 1 L sterile growth medium. The growth medium was composed of the following nutrients: potassium sulfate 18.2 g/L, ammonium phosphate monobasic 36.4 g/L, potassium phosphate dibasic 12.8 g/L, magnesium sulfate heptahydrate 3.72 g/L, sodium citrate dihydrate 10 g/L, glycerol 40 g/L, yeast extract 30 g/L, PTM1 trace metals 4.35 mL/L, and antifoam 204 1.67 mL/L. The PTM1 trace metal solution was comprised of the following components: cupric sulfate pentahydrate 6 g/L, sodium iodide 226 2016273912 14 Dec 2016 0.08 g/L, manganese sulfate hydrate 3 g/L, sodium molybdate dihyrate 0.2 g/L, boric acid 0.02 g/L, cobalt chloride 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 g/L, biotin 0.2 g/L, and sulfuric acid 5 mL/L. The yeast strain was engineered to express Ab-A antibody from four integrated genomic copies of the heavy chain coding sequence (SEQ ID NO: 441) and 3 copies of the light chain coding sequence (SEQ ID NO: 440). The heavy chain gene copies were integrated into the pGAP locus (3 copies) and HIS4 TT locus (1 copy) while the 3 light chain gene copies were integrated into the pGAP locus. The antibody chain gene copies were each under the control of the GAP promoter. The bioreactor process control parameters were set as follows: Agitation 1000 rpm, airflow 1.35 standard liter per minute, temperature 28°C and pH was controlled (at 6) using ammonium hydroxide. No oxygen supplementation was provided.

[00879] Following addition of the inoculum, fermentation cultures were grown for approximately 12 to 16 hours (the “growth phase”). The growth phase ended when the initial glycerol in the medium was consumed, as which was detected by a dissolved oxygen (“DO”) spike (a sudden increase in the dissolved oxygen concentration). The cultures were then starved for approximately three hours after the dissolved oxygen spike (“starvation phase”) for run L306. For other runs, the ethanol bolus was added immediately after DO spike. A bolus of ethanol was then added to the reactor to give a final concentration of 1% ethanol (w/v). Control cultures were treated identically, except that the bolus addition of ethanol was omitted. The fermentation cultures were allowed to equilibrate for 15 to 30 minutes (“equilibration phase”). After the equilibration phase, feed was added at a constant rate of 30 g/L/hr for 40 minutes (“transition phase”). For the remainder of the culture (“production phase”) the ethanol concentration was detected using an ethanol sensing probe (Raven Biotech) which was used to control the feed rate, with the feed rate being set at 15 g/L/hr when the ethanol concentration was below the set point, or 7.5 g/L/hr when the ethanol concentration was above the set point. In instances in which the high feed rate of 15 g/L/hr was not high enough to maintain ethanol at set point (which occurred in the L315 fermentation run), the high feed rate was set to 22.5 g/L/hr while the low feed rate was set to 15 g/L/hr. The same set point was maintained whether or not an ethanol bolus had been added to the culture (production of ethanol by the yeast caused the set point to be reached without the bolus addition of ethanol). The feed was composed of the following components: yeast extract 50 g/L, anhydrous dextrose 500 g/L, magnesium sulfate heptahydrate 3 g/L, PTM1 trace metals 12 mL/L, and sodium citrate dihydrate 0.5g/L The total fermentation time was 227 2016273912 14 Dec 2016 typically 85 hours to 97 hours in these experiments, though longer and shorter times can also be used.

[00880] After the production phase, fermentation cultures had PEI (polyethyleneimine) and EDTA (ethylenediaminetetraacetic acid) added to 0.05% w/v and 3 mM final concentrations respectively. The cultures were then spun in a centrifuge and antibodies were purified from the culture supernatant by Protein A affinity. Briefly, approximately 20 mL of 0.2μ clarified supernatants from harvested fermentation broth were diluted with the same volume of equilibration buffer (20 mM Histidine pH6). From this diluted broth, 20 mL were then loaded onto a pre-equilibrated 1 mL HiTrap MabSelect Sure column (GE, Piscataway, NJ). The column was subsequently washed using 40 column volumes of equilibration buffer. The antibody bound onto the column was eluted using a step gradient into 100% elution buffer (100 mM Citric Acid pH 3.0). One mL fractions were collected and immediately neutralized using lOOqL of 2M Tris buffer pH 8.0. Protein containing fractions were determined by measuring absorbance at 280nM and protein-containing fractions were pooled.

[00881] Protein A purified antibodies were analyzed for purity by SDS-PAGE. For non-reduced samples, SDS-PAGE was carried out using precast polyacrylamide gels (NuPAGE® Bis-Tris Gels) containing a 4%-12% polyacrylamide gradient, using NuPAGE® MES SDS running buffer and NuPAGE® LDS Sample Buffer (all from Invitrogen, Carlsbad, Ca.) in accord with the manufacturer’s instructions. Proteins were then visualized by Coomassie blue staining. Reduced samples were processed in the same manner except that samples were reduced prior to loading using the NuPAGE® Sample Reducing Agent (Invitrogen, Carlsbad, Ca.) in accord with the manufacturer’s instructions.

[00882] Results [00883] To determine the effect of a bolus addition of ethanol during culture on antibody purity, the antibody Ab-A was produced from yeast cultures with or without the addition of a bolus of ethanol to a final concentration of 1% (10 g/L) at the end of the growth phase and prior to the production phase. The production phase was continued for 97 hours (FIG. 1), 87 hours (FIG. 2), or 86 hours (FIG. 3). The antibody produced by each culture was then harvested from the culture media, purified by Protein A affinity chromatography. 228 2016273912 14 Dec 2016 [00884] SDS-PAGE was used to detect the relative abundance of the full antibody, the “half antibody” or H1L1 complex (containing one heavy and one light chain) and the H2L1 complex (containing two heavy chains and one light chain). The abundance of the H1L1 and H2L1 complexes was greatly decreased in the cultures that were produced with the bolus addition of ethanol. This improvement was reproduced in three experiments shown in FIG. 1 A, compare lanes 2 and 3 (with bolus) to 5 (no bolus); FIG. 2A, compare lane 2 (with bolus) to 3 (no bolus); FIG. 3A, compare lanes 2 and 4 (with bolus) to lanes 5-7 (no bolus). Under reducing conditions, the H1L1, H2L1, and full antibody species were each separated into individual heavy and light chains, confirming the identity of the 75 kDa band as consisting of one light and one heavy chain joined by a disulfide-linkage (FIGS. IB and 2B; lane order is the same as FIGS. 1A and IB, respectively).

[00885] The decrease in abundance of the H1L1 species was then quantified using Image J to plot the gel band density along the length of the non-reduced gels (FIGS. 1C-E and 2C-D, respectively corresponding to FIG. 1A, lanes 2, 3, and 5, and FIG. 2A, lanes 2 and 3, and FIG. 3B, corresponding to lanes 2 and 4-6 of FIG. 3A). The area under the H1L1 peak was quantified and results are tabulated in FIGS. IF, 2E, and 3B. Based on these measurements, the addition of an ethanol bolus prior to antibody production decreased the relative abundance of the 75 kDa band by about 90% in FIG. 1 A, by about 85% in FIG. 2A, and by about 87% in FIG. 3A.

[00886] In summary, these results demonstrate that the concentration of the HI El species was greatly decreased by the bolus addition of ethanol to the culture, resulting in decreased production of the H1L1 species between about 85% to 90%.

[00887] EXAMPLE 2 [00888] This example extends the results obtained in Example 1 by demonstrating that the same methods produced a similar improvement in antibody purity when used during production of two additional antibodies.

[00889] Methods [00890] Antibodies Ab-B and Ab-C were recombinantly produced using the methods described in Example 1. Antibody Ab-C was expressed from a yeast strain engineered contain 229 2016273912 14 Dec 2016 four copies of the heavy chain coding sequence (SEQ ID NO: 439) and three copies of the light chain coding sequence (SEQ ID NO: 438). Samples were taken from the reactors and culture supernatant containing antibodies was collected after a total fermentation time of 67 hours (T67) or 87 hours (T87) for the Ab-B antibody and for 86 hours (T86) for the Ab-C antibody, purified by Protein A affinity, and analyzed by SDS-PAGE as described in Example 1.

[00891] Results [00892] Antibodies Ab-B (FIG. 4) and Ab-C (FIG. 5) were produced with or without a bolus addition of ethanol to a final concentration of 1% at the end of the growth phase and prior to the production phase. For the antibodies produced without the bolus addition of ethanol, the H1L1 or half antibody species, and the H2L1 species were each observed as a prominent band (FIG. 4A, lines 6 and 7; FIG. 4C, lanes 6 and 7; FIG. 5A, lanes 5 and 6). The intensity of these bands were greatly decreased for the culture produced with a bolus addition of ethanol (FIG. 4A, lanes 2-3; FIG. 4C, lanes 2-3; FIG. 5 A, lane 3). Under reducing conditions, the H1L1 and H2L1 bands were separated into individual heavy and light chains, confirming the identity of these species as consisting full length heavy and light chains (FIGS. 4B, 4D, and 5B; lane order is the same as in FIGS. 4A, 4D, and 5A, respectively).

[00893] The decrease in abundance of the H1F1 species was then quantified using Image J to plot the gel band density along the length of the non-reduced gels. FIGS. 4E and 4F tabulate the area contained in the H1F1 peaks shown in FIGS. 4A (T67) and 4C (T87), respectively, demonstrating that the bolus addition of ethanol produced about a 73% reduction in the relative abundance of H1F1 complexes at the earlier time point shown FIG. 4A and about a 34% average reduction in the relative abundance of H1F1 complexes at the later time point shown in FIG. 4C. Similarly, FIG. 5C tabulates the area contained in the HI LI peaks shown in FIGS. 5 A, demonstrating about a 61% average reduction in the relative abundance of H1L1 complexes by the bolus addition of ethanol.

[00894] In summary, these results demonstrate that the concentration of the H1F1 and H2L1 species were decreased by between about 61% and 73% by the bolus addition of ethanol to the culture for two additional antibodies having binding specificity for different targets. 230 2016273912 14 Dec 2016 [00895] EXAMPLE 3 [00896] This example further describes the improved purity of recombinant antibodies produced from cultured P. pastoris cells through addition of a bolus of ethanol during culturing. In addition to greatly decreasing the abundance of the H1L1 and H2L1 species, this example further demonstrates a decrease in the concentration of other product-associated variants.

[00897] Methods [00898] Recombinant antibodies Ab-A, Ab-B, and Ab-C were prepared and purified from P. pastoris cultures as described in Examples 1 and 2. Antibodies were produced either with or without a bolus addition of ethanol to a final concentration of 1% (w/v) at the end of the growth phase and prior to the production phase. To analyze the purity of protein A purified antibody preparations, size exclusion high-performance liquid chromatography (SE-HPLC) was used. Briefly, an Agilent (Santa Clara, CA) 1200 Series HPLC with UV detection instrument was used. For sample separation, a TSKgel 3000SWxl 7.8x300 mm column connected with a TSKgel Guard SWxl 6x40 mm from Tosoh Bioscience (King of Prussia, PA) was used. A solution of 100 mM sodium phosphate, 200 mM sodium chloride pH 6.5 was used as mobile phase with a flow rate of 0.5 mL/min in isocratic mode and absorbance at UV 215nm was monitored. Before injection of samples the column was equilibrated until a stable baseline was achieved. Samples were diluted to a concentration of 1 mg/mL using mobile phase and a 30 pL volume was injected. To monitor column performance, BioRad (Hercules, CA) gel filtration standards were used.

[00899] Results [00900] The Ab-A antibody preparations described in Example 1 and additional preparations produced using the same methods were expressed in yeast, purified by protein A affinity, and then analyzed for purity using size exclusion chromatography (SEC). Under the condition used, the half antibody (H1L1) species co-elutes with the full antibody, which is thought to be due to non-covalent association between pairs of half antibodies. However, this method allows purity to be assessed with respect to other product-associated variants, such complexes having aberrant stoichiometry, fragments, glycosylated forms, and aggregates. 231 2016273912 14 Dec 2016 [00901] SE-HPLC data are shown for Ab-A samples produced without (FIGS. 6A, 6C, and 6E) or with (FIGS. 6B, 6D, and 6F) a bolus addition of ethanol. A product-associated variant consisting of an aggregate of two full antibodies (containing four heavy and four light chains) was detected (arrow), and the abundance thereof was reduced on average in the samples prepared with the bolus addition. FIG. 7 shows quantification of the purity of Ab-A by determining the percentage of the antibody preparation contained in the main peak (containing the full antibody). The bolus addition of ethanol increased the average percentage contained in the main peak from 80.3% up to 90.6%.

[00902] Similar analysis was performed for the Ab-B and Ab-C antibody preparations described in Example 2, quantified in FIGS. 8 and 9, respectively. Overall purity of the Ab-B antibody was improved, with the average fraction in the main peak increasing from 76% to 79% at T67 and from 60% to 73% at T87. For the Ab-C antibody, there was little detectable difference in antibody purity assessed by this method, apparently due to the high initial purity of the Ab-C antibody even without the bolus addition.

[00903] In summary, these results demonstrate that a bolus addition of ethanol to the culture can decrease the concentration of other product-associated variants in addition to the half-antibody species.

[00904] EXAMPLE 4 [00905] This example describes the further confirmation of the identity of the 75 kDa product associated variant as a half antibody species containing only one antibody heavy chain and only one antibody light chain. This hypothesis was based on several observations. First, the 75 kDa band was present in protein A purified samples (see Example 1) indicating that it contained at least the protein A binding portion of an antibody heavy chain. Second, the 75 kDa band was prominent in non-reduced samples analyzed by SDS-PAGE (see, e.g., FIG. 1 A, lanes

2-3), but under reducing conditions the same samples did not contain any bands of comparable intensity (other than the expected light and heavy chains), indicating that the 75 kDa band does not include any components other than antibody heavy and light chains (or other species having the same electrophoretic mobility). Third, disappearance of the 75 kDa band from the reduced samples also indicates that its constituents are linked by at least one disulfide bond. Fourth, SEC 232 2016273912 14 Dec 2016 analysis had demonstrated co-elution of the 75 kDa species with the full antibody, strongly suggesting that the 75 kDa species can non-covalently self-associate to form a full antibody (or another complex of the same apparent hydrodynamic radius). Finally, the apparent molecular weight of about 75 kDa (determined by reference to electrophoresis standards), taken together with the observation (from denaturing gels) that this complex was only made up of full-length antibody chains, was consistent with complex containing only one heavy chain and only one light chain, but was inconsistent with other complexes.

[00906] Methods [00907] Mass spectrometry was used to detect the relative abundance of heavy chains lacking the inter-heavy chain disulfide bonds (normally found at amino acids 220 and 223) in different samples. Two-hundred and fifty micrograms of each sample was added into an Eppendorf tube. An appropriate amount (~450 pL) of denaturing buffer (6 M Guanidine-HCl, 1 mM EDTA, 0.25 M Tris, pH 7.5) was added to the tube to obtain a final volume of 500 pL and a sample concentration of 0.5 mg/mL. Twelve and a half microliters of 2 M Iodoacetamide was added into each sample to alkylate any free cysteine. The samples were vortexed then incubated at room temperature, in the dark, for 30 ± 5 minutes. The samples were then desalted using NAP-5 columns pre-equilibrated with digestion buffer (0.1 M Tris-HCl, pH 7.5). Each sample solution was added to separate (pre-equilibrated) columns and allowed to enter the column bed. One milliliter of digestion buffer was added to each column and the eluent was collected into Eppendorf tubes. The samples were divided into equal aliquots containing approximately 50 pg of material (five 200 pL aliquots). The alkylated and desalted aliquots were stored at -20 °C until needed. One aliquot of each sample (alkylated, and desalted) was used for each digestion. Trypsin solution at 0.5 mg/mL was added to each sample aliquot at a 1:25 w:w ratio of trypsimprotein (4 pL). All trypsin tubes were incubated at 37 ± 2 °C for 4 hours. After incubation, the enzymatic digestion was quenched by adding 1 pL of Trifluoroacetic acid to each tube. The samples were then divided into two equal portions, reduced and non-reduced. Half the samples were reduced in the presence of 1 M DTT for 1 hour at 37 ± 2 °C. The contents of both reduced and nonreduced samples were transferred to HPLC vials and placed in the autosampler for analysis. 233 2016273912 14 Dec 2016 [00908] MS and MS/MS data was collected on a Micromass Q-TOF Ultima mass spectrometer using electrospray ionization (ESI) in positive ion mode. Data was acquired from m/z 200-1950 in MS mode. Prior to analysis, the mass spectrometer was calibrated using a 5th order fit on fragment ions of [Glu']-Fibrinopeptide covering a range from m/z 175 to 1285. The injections volumes were adjusted to achieve an on-column load of approximately 20 pmoles of protein.

[00909] Results [00910] Based on the observations discussed above, Applicants hypothesized that the 75 kDa band was a “half antibody” species containing one heavy antibody chain and one light antibody chain covalently linked to one another through disulfide bonds, and that pairs of half antibody complexes could non-covalently associate to form a complex having the same stoichiometry as a full antibody (two heavy chains and two light chains) but lacking the disulfide linkages between the two heavy chains (“unlinked heavy chains”). Based thereon, it was predicted the relative abundance of the 75 kDa band would correlate with the relative proportion of unlinked heavy chains, which was determined using mass spectrometry analysis of trypsin digested antibody samples.

[00911] The peptide fragments of interest for this study were the T17 trypsin fragment of the heavy chain (T17H) which is composed of amino acids 217-242, respectively. Amino acids 220 and 223 are responsible for disulfide bonding between antibody heavy chains. Typically, free cysteine analysis can be conducted by determining a ratio of alkylated cysteine residues to non-alkylated residues in reduced samples after a tryptic digestion. However, alkylated species were not present in either lot of material. It was hypothesized the two cysteine residues of the peptide fragment of interest, T17H, were bonding to each other, or the cysteines were protected by the antibody’s quaternary structure. In either case, the cysteine residues would not be accessible for alkylation. Instead, the analytical approach was to utilize the ratio of non-reduced species to reduced species in both lots to calculate a percent difference. It was observed that the non-reduced samples had a 2 Da decrease in molecular weight, indicative of disulfide bonding. This behavior was exploited to calculate the percent free T17H. The theoretical mass of the non-234 2016273912 14 Dec 2016 reduced T17H species is 2727.41 Da (disulfide bonding between Cys220 & Cys223) and 2729.41 Da for reduced peptide.

[00912] The extracted ion chromatograms of reduced and non-reduced samples were analyzed for representative charge states, ratio of counts between the non-reduced to reduced samples calculates the free T17H species to be 2.3% in the antibody produced with a bolus addition of ethanol, and 26.1% in the sample produced without a bolus addition of ethanol.

These results are presented in tabular form in Fig. 9.

[00913] Thus, the abundance of heavy chains lacking the inter-heavy chain disulfide bond was greatly increased in the sample produced without the bolus addition of ethanol, further confirming the identity of this species as containing one heavy chain and one light chain but lacking an inter-heavy chain disulfide bond. Moreover, the detection of a species 2 Da lighter than the expected mass indicated that heavy chain in the H1L1 species may contain an extra disulfide bond to itself which may interfere with the formation of the normal inter-heavy chain disulfide bond.

[00914] EXAMPLE 5 [00915] This example demonstrates a correlation between cell viability and antibody purity. The addition of an ethanol bolus generally improved cell viability and antibody purity for Ab-A and the Ab-B antibody. Further, the Ab-C antibody, which already exhibited high purity even without the bolus addition, also exhibited higher culture viability. Taken together, these results suggest that the improvement in antibody purity resulting from a bolus addition of ethanol is at least partially attributable to increased culture viability.

[00916] Methods [00917] Culture viability was determined using a Cellometer (Nexcelom). Culture samples were diluted with PBS so that the final cell count was within 1 x 107 to 5 x 107 cells/mL. One half of the sample was then treated to heat conditions of 75°C for 10 minutes as positive control for propidium iodide (PI) staining. The untreated sample and treated sample were then mixed with PI (20 uL of sample plus 20 uL of PI). The sample was then placed in a slide cassette and viability was determined by counting the number of non-fluorescing cells then dividing by the 235 2016273912 14 Dec 2016 total number of cells. Cells that are dead have taken up the propidium iodide and are fluorescing so the positive control heat killed sample should show less than 1% cells alive.

[00918] Results [00919] Cell viability was determined for antibody-producing cultures grown with or without a bolus addition of ethanol as described in Examples 1 and 2. As described above, purity of the Ab-A and Ab-B antibodies were greatly improved by a bolus addition of ethanol to the yeast culture (see Examples 1-2 and FIGS. 1-4). The addition of an ethanol bolus improved cell viability for these cultures as well. For the Ab-A antibody, viability improved from 91.9% to 97.2% on average (FIG. 11), while for the Ab-B antibody, viability improved from 84.8% to 95.1% on average (FIG. 12). Due to the already high purity of the Ab-C antibody produced even without the bolus addition of ethanol, the improvements in the purity of this antibody resulting from the bolus addition of ethanol were more modest (see Example 3 and FIG. 5). Consistent with the observation that high cell viability correlated with higher antibody purity, the Ab-C antibody cultures exhibited high cell viability (95.8%% on average) in the absence of a bolus addition of ethanol, which was little changed by the bolus addition of ethanol (96.8%).

[00920] Taken together, these results indicate that the improvement in antibody purity resulting from the addition of an ethanol bolus may be in part caused by (or at least correlates with) an improvement in cell viability.

[00921] EXAMPLE 6 [00922] This example demonstrates that a similar improvement in antibody purity can be attained with varying ethanol bolus concentrations.

[00923] Methods [00924] The Ab-A antibody was produced as in Example 1, except that the bolus addition of ethanol was 5 g/L (0.5% w/v), 10 g/L (1% w/v), or 15 g/L (1.5% w/v). Antibodies samples were purified from the culture media at 63 and 86 hours and purified by protein A affinity, then the purity was analyzed by non-reduced SDS-PAGE as in Example 1. 236 2016273912 14 Dec 2016 [00925] Results [00926] Antibody purity was similarly high irrespective of the bolus concentration added (between 0.5% and 1.5% w/v), at both time points tested, 63 hours (FIG. 14A) and 86 hours (FIG, 14B). Detected levels of the H1L1 and H2L1 species were similarly low in each culture.

[00927] These results indicate that the improvement in antibody purity can be attained while varying the ethanol bolus concentration.

[00928] EXAMPLE 7 [00929] This example demonstrates that similar improvements in antibody purity can be attained while varying the duration of the “starvation period” between the dissolved oxygen spike and the addition of the ethanol bolus to the cultures.

[00930] Methods [00931] The Ab-A antibody was produced as in Example 1, except that the duration of the starvation period, the time between the dissolved oxygen spike (indicating exhaustion of the carbon source in the culture) and the bolus addition of ethanol, was either 0 hours or 3 hours. Antibodies samples were purified from the culture media and purified by protein A affinity, then the purity was analyzed by non-reduced SDS-PAGE as in Example 1.

[00932] Results [00933] Antibody purity was similarly high irrespective of the variation of the starvation period between 0 and 3 hours (FIG. 15 A, compare lanes 5 (0 hours starvation) and 6 (3 hours starvation)). Detected levels of the H1L1 and H2L1 species were similarly low in each culture.

[00934] These results indicate that the improvement in antibody purity can be attained with a varying the duration of the starvation period. 237 2016273912 14 Dec 2016 [00935] EXAMPLE 8 [00936] This example tests the effect on antibody purity from varying the duration of the “equilibration period” between the addition of the ethanol bolus and commencement of adding the feed to the cultures.

[00937] Methods [00938] The Ab-B antibody was produced as in Example 2, except that the duration of the equilibration period, the time between addition of the ethanol bolus and the commencement of feeding the culture, was 0, 30, or 60 minutes. Additionally, the yeast strain from which the Ab-B antibody was produced contained three copies of the light chain gene instead of four. Antibodies samples were purified from the culture media and purified by protein A affinity, then the purity was analyzed by non-reduced SDS-PAGE as in Example 1. Viability was also assessed using the methods described in Example 5.

[00939] Results [00940] Antibody purity was similarly high for an equilibration period of 0 or 30 minutes (FIG. 16A, lanes 7 and 8 (0 minutes equilibration time) and lane 3 (30 minutes equilibration time). However, detected levels of the H1L1 and H2L1 species were increased in the culture with the 60 minute equilibration period (FIG. 16A, lanes 5 and 6).

[00941] Viability was also assessed for each culture at the 23 hour and 85 hour time points. For the 60 minute equilibration period, viability was between 75% and 80% at 23 hours, while at the same time point viability was approximately 88-90% for the 0 and 30 minute equilibration periods (FIG. 16B). Subsequently, at 85 hours, viability had improved but remained somewhat reduced for the 60 minute equilibration period relative to the 0 and 30 minute equilibration periods (FIG. 16C).

[00942] These results indicate that the improvement in antibody purity can be attained while varying the equilibration period at least between 0 and 30 minutes, while some loss of viability and purity may occur for an equilibration period of 60 minutes or longer (though purity may still be improved relative to a control culture without a bolus addition of ethanol). 238 2016273912 14 Dec 2016 [00943] The above description of various illustrated embodiments of the invention is not intended to be exhaustive or to limit the invention to the precise form disclosed. While specific embodiments of, and examples for, the invention are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the invention, as those skilled in the relevant art will recognize. The teachings provided herein of the invention can be applied to other purposes, other than the examples described above.

[00944] The invention may be practiced in ways other than those particularly described in the foregoing description and examples. Numerous modifications and variations of the invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

[00945] These and other changes can be made to the invention in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the invention to the specific embodiments disclosed in the specification and the claims. Accordingly, the invention is not limited by the disclosure, but instead the scope of the invention is to be determined entirely by the following claims.

[00946] Certain teachings related to methods for obtaining a clonal population of antigen-specific B cells were disclosed in U.S. Provisional patent application no. 60/801,412, filed May 19, 2006, the disclosure of which is herein incorporated by reference in its entirety.

[00947] Certain teachings related to humanization of rabbit-derived monoclonal antibodies and preferred sequence modifications to maintain antigen binding affinity were disclosed in International Application No. PCT/US2008/064421, corresponding to International Publication No. WO/2008/144757, entitled “Novel Rabbit Antibody Humanization Methods and Humanized Rabbit Antibodies”, filed May 21, 2008, the disclosure of which is herein incorporated by reference in its entirety.

[00948] Certain teachings related to producing antibodies or fragments thereof using mating competent yeast and corresponding methods were disclosed in U.S. Patent application no. 239 2016273912 14 Dec 2016 11/429,053, filed May 8, 2006, (U.S. Patent Application Publication No. US2006/0270045), the disclosure of which is herein incorporated by reference in its entirety.

[00949] The entire disclosure of each document cited herein (including patents, patent applications, journal articles, abstracts, manuals, books, or other disclosures), including each document cited in the Background, Summary, Detailed Description, and Examples, is hereby incorporated by reference herein in its entirety. 240

Claims (105)

1. Claims

   1. A method of producing a multi-subunit complex, comprising: (a) providing a culture comprising a eukaryotic cells comprising genes that provide for the expression of the subunits of said multi-subunit complex; (b) adding a bolus of ethanol to said culture; and (c) culturing said culture to produce said multi-subunit complex.
2. 2. The method of claim 1, wherein the ethanol bolus enhances the formation of stable disulfide bonds relative to the same method effected in the absence of the bolus of ethanol.
3. 3. The method of claim 1, wherein said multi-subunit complex contains one or more polypeptides comprising at least one disulfide bond.
4. 4. The method of claim 1, wherein said multi-subunit complex comprises an antibody.
5. 5. The method of any one of claims 1 to 4, which decreases the relative abundance of one or more product-associated variants relative to the same method effected in the absence of the bolus of ethanol.
6. 6. The method of any one of claims 1 to 4, which decreases the relative abundance of product-associated variants having a higher or lower apparent molecular weight than said desired multi-subunit complex as detected by size exclusion chromatography or gel electrophoresis relative to the same method effected in the absence of the bolus of ethanol.
7. 7. The method of any one of claims 1 to 4, which decreases the relative abundance of complexes having aberrant stoichiometry relative to the same method effected in the absence of the bolus of ethanol.
8. 8. The method of any one of claims 1 to 4, which decreases the relative abundance of complexes having aberrant disulfide bonds relative to the same method effected in the absence of the bolus of ethanol.
9. 9. The method of any one of claims 1 to 4, which decreases the relative abundance of complexes having reduced cysteines relative to the same method effected in the absence of the bolus of ethanol.
10. 10. The method of any one of claims 1 to 4, which decreases the relative abundance of complexes having aberrant glycosylation relative to the same method effected in the absence of the bolus of ethanol.
11. 11. The method of claim 4, which modulates the formation or stability of inter-heavy chain disulfide bonds.
12. 12. The method of claim 4 or 11, which modulates the formation or stability of disulfide bonds linking the light and heavy chains.
13. 13. The method of any one of claims 4 or 11 to 12, which decreases the relative abundance of one or more product-associated variants relative to the same method effected in the absence of the bolus of ethanol.
14. 14. The method of claim 13, wherein said product-associated variants comprise one or more of the H1L1, H2L1, and H4L4 product-associate variants.
15. 15. The method of any one of claims 4 or 11 to 14, which increases the purity of said antibody relative to said method effected in the absence of said bolus of ethanol.
16. 16. The method of any one of the foregoing claims, wherein step (b) is effected prior to step (c).
17. 17. The method of any one of claims 1 to 15, wherein step (b) is effected subsequent to step (c).
18. 18. The method of any one of claims 1 to 15, wherein step (b) is effected concurrently with step (c).
19. 19. The method of any one of the foregoing claims, wherein step (b) results in a concentration of ethanol in said culture of between about 0.01% and about 4% (w/v).
20. 20. The method of any one of the foregoing claims, wherein step (b) results in a concentration of ethanol in said culture of between about 0.01% and about 4%, between about 0.02% and about 3.75%, between about 0.04% and about 3.5%, between about 0.08% and about 3.25%, between about 0.1% and about 3%, between about 0.2% and about 2.75%, between about 0.3% and about 2.5%, between about 0.4% and about 2.25%, between about 0.5% and about 1.5%, between about 0.5% and about 2%, between about 0.6% and about 1.75%, between about 0.7% and about 1.5%, or between about 0.8% and about 1.25%.
21. 21. The method of any one of the foregoing claims, wherein step (b) results in a concentration of ethanol in said culture that is at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.6%, 0.6%, 0.7%, 0.8% or 0.9% (w/v).
22. 22. The method of any one of the foregoing claims, wherein step (b) results in a concentration of ethanol in said culture that is at most about 4%, 3.5%, 3%, 2.5%, 2%, 1.8%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, or 0.15% (w/v).
23. 23. The method of any one of the foregoing claims, wherein step (b) comprises adding ethanol to said culture, adding a carrier comprising ethanol to said culture, adding said cells to a medium or carrier comprising ethanol, or replacing part of the culture medium.
24. 24. The method of any one of the foregoing claims, wherein said bolus of ethanol is added to the culture medium over a period of time between 1 and 20 minutes.
25. 25. The method of any one of the foregoing claims, wherein step (c) comprises providing oxygen to said cells.
26. 26. The method of claim 25, wherein said providing oxygen comprises agitating said culture.
27. 27. The method of claim 25 or 26, wherein said providing oxygen comprises contacting said culture with a gas mixture comprising oxygen.
28. 28. The method of any one of the foregoing claims, wherein step (c) comprises adding a feed comprising a carbon source to said culture.
29. 29. The method of claim 28, wherein said feed comprises at least one fermentable carbon source.
30. 30. The method of claim 28, wherein said feed comprises one or more of glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose, mannitol, and raffinose.
31. 31. The method of any one of the foregoing claims, further comprising maintaining the concentration of ethanol between an upper set point and a lower set point during step (c).
32. 32. The method of claim 31, wherein said lower set point is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.6%, 0.6%, 0.7%, 0.8% or 0.9% (w/v).
33. 33. The method of claim 31 or 32, wherein said upper set point is about 4%, 3.5%, 3%, 2.5%, 2%, 1.8%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, or 0.15% (w/v).
34. 34. The method of claim 31 or 32, wherein said upper set point is at most about 1.5%, 1.4%, 1.3, 1.2%, or 1.1% (w/v).
35. 35. The method of any one of claims 1 to 30, further comprising maintaining the concentration of ethanol at a set point during step (c).
36. 36. The method of claim 35, wherein said set point is about 0.1%, 0.2%, 0. 3%, 0. 4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 01.%, 01.1%, 01.2%, 01.3%, 01.4%, or 01.5% (w/v).
37. 37. The method of any one of claims 1 to 30, wherein step (c) comprises maintaining the concentration of ethanol in said culture between about 0.01% and about 4%, between about 0.02% and about 3.75%, between about 0.04% and about 3.5%, between about 0.08% and about 3.25%, between about 0.1% and about 3%, between about 0.2% and about 2.75%, between about 0.3% and about 2.5%, between about 0.4% and about 2.25%, between about 0.5% and about 2%, between about 0.6% and about 1.75%, between about 0.7% and about 1.5%, or between about 0.8% and about 1.25%.
38. 38. The method of any one of claims 21 to 27, wherein the concentration of ethanol in said culture is maintained by controlling production of ethanol by said cells or by addition of ethanol to said culture.
39. 39. The method of claim 28, wherein the step of controlling production of ethanol comprises controlling one or more of the concentration of glucose, availability of oxygen, intensity of agitation, gas pressure, flow rate of supplied air or other gas mixture, viscosity of the culture, culture density, concentration of oxygen in the supplied air or other gas mixture, and temperature.
40. 40. The method of any one of the foregoing claims, wherein the time between step (a) and step (b) is less than about 72 hours, less than about 48 hours, less than about 24 hours, less than about 12 hours, less than about 9 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 90 minutes, less than about 30 minutes, less than about 5 minutes, or less than about 1 minute.
41. 41. The method of any one of the foregoing claims, wherein the time between step (b) and step (c) is less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, less than about 90 minutes, less than about 80 minutes, less than about 70 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 30 minutes, less than about 20 minutes, less than about 10 minutes, less than about 5 minutes, or less than about 1 minute.
42. 42. The method of any one of the foregoing claims, wherein the culture of step (a) is produced by adding a carbon source to said culture, and culturing said culture until the carbon source is depleted.
43. 43. The method of claim 42, wherein said carbon source comprises one or more of: glycerol, glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose, mannitol, and raffinose.
44. 44. The method of claim 42 or 43, wherein the depletion of the carbon source is determined by detecting a decrease in the metabolic activity of said eukaryotic cells.
45. 45. The method of claim 44, wherein said decrease in the metabolic activity of said eukaryotic cells is identified by detecting a decrease in the consumption of oxygen by said eukaryotic cells, by detecting an increase in pH in the culture, by detecting stabilization of the wet cell mass, or by detecting an increase in the concentration of ammonia in the culture.
46. 46. The method of claim 45, wherein said decrease in the consumption of oxygen by said eukaryotic cells is identified by detecting an increase in the concentration of dissolved oxygen in said culture.
47. 47. The method of any one of the foregoing claims, wherein said eukaryotic cells comprise yeast cells.
48. 48. The method of claim 47, wherein said yeast cells comprise methylotrophic yeast.
49. 49. The method of claim 48, wherein said methylotrophic yeast is of the genus Pichia.
50. 50. The method of claim 49, wherein said methylotrophic yeast of the genus Pichia is Pichia pastoris.
51. 51. The method of claim 49, wherein said methylotrophic yeast of the genus Pichia is selected from the group consisting of: Pichia angusta, Pichia guillermordii, Pichia methanolica, and Pichia inositovera.
52. 52. The method of claim 49, wherein the genes that provide for expression of said multisubunit complex are integrated into one or more genomic loci.
53. 53. The method of claim 49, wherein at least one of said genomic loci are selected from the group consisting of the pGAP locus, 3’ AOX TT locus; PpURA5; OCH1; AOX1; HIS4; GAP; pGAP; 3’ AOX TT; ARG; and the HIS4 TT locus.
54. 54. The method of any one of the foregoing claims, wherein at least one of the genes encoding said subunits of the multi-subunit complex are expressed under control of an inducible or constitutive promoter.
55. 55. The method of claim 54, wherein said inducible promoter is selected from the group consisting of the AOX1, CUP1, tetracycline inducible, thiamine inducible, and FLD1 promoters.
56. 56. The method of any one of the foregoing claims, wherein at least one of the genes encoding said subunits of the multi-subunit complex are expressed under control of a promoter selected from the group consisting of: the CUP1, AOX1, ICL1, glyceraldehyde-3-phosphate dehydrogenase (GAP), FLD1, ADH1, alcohol dehydrogenase II, GAL4, PH03, PH05, and Pyk promoters, tetracycline inducible promoters, thiamine inducible promoters, chimeric promoters derived therefrom, yeast promoters, mammalian promoters, insect promoters, plant promoters, reptile promoters, amphibian promoters, viral promoters, and avian promoters.
57. 57. The method of any one of the foregoing claims, wherein said eukaryotic cell is a diploid, tetraploid cell, or polyploid.
58. 58. The method of any one of the foregoing claims, further comprising purifying said multisubunit complex from said eukaryotic cells or from the culture medium.
59. 59. The method of claim 58, wherein said multi-subunit complex is purified from an intracellular component, cytoplasm, nucleoplasm, or a membrane of said eukaryotic cells.
60. 60. The method of claim 58, wherein said eukaryotic cells secrete said multi-subunit complex into the culture medium.
61. 61. The method of claim 60, wherein said multi-subunit complex is purified from said culture medium.
62. 62. The method of any one of the foregoing claims, wherein said multi-subunit complex comprises a monospecific or bispecific antibody.
63. 63. The method of any one of the foregoing claims, wherein said multi-subunit complex comprises a human antibody or a humanized antibody or fragment thereof.
64. 64. The method of claim 63, wherein said humanized antibody is of mouse, rat, rabbit, goat, sheep, or cow origin.
65. 65. The method of claim 62, wherein said humanized antibody is of rabbit origin.
66. 66. The method of any one of the foregoing claims, wherein said multi-subunit complex comprises a monovalent, bivalent, or multivalent antibody.
67. 67. The method of claim 4 or any one of claims 61 to 66, wherein said antibody is purified from said culture by protein A and/or protein G affinity.
68. 68. The method of any one of the foregoing claims, wherein at least one of the genes that provide for expression of a subunit of said multi-subunit complex in at least one of said eukaryotic cells in said panel is optimized for expression in said eukaryotic cell.
69. 69. The method of any one of the foregoing claims, wherein said multi-subunit complex comprises an antibody and the purity of said antibody is assessed by measuring the fraction of the antibody produced by said eukaryotic cell that is contained in antibody complexes having the expected apparent hydrodynamic radius, is contained in antibody complexes having the expected molecular weight, and / or specifically binds a target of said antibody.
70. 70. The method of any one of the foregoing claims, wherein said multi-subunit complex comprises an antibody and the yield of said antibody is assessed by determining the amount of antibody produced by said eukaryotic cell discounting any product-associated variants that are abnormally glycosylated, contained in antibody complexes other than complexes having the expected apparent hydrodynamic radius, contained in antibody complexes having the expected molecular weight, and / or that fail to specifically bind to the target of said antibody.
71. 71. The method of claim 58 or 59, wherein the molecular weight of said antibody complexes is determined by non-reducing SDS-PAGE.
72. 72. The method of any one of the foregoing claims, wherein said multi-subunit complex comprises an antibody, said method further comprising purifying said antibody.
73. 73. The method of any one of the foregoing claims, wherein said culture cell produces a supernatant antibody titer of at least 100 mg / L, at least 150 mg / L, at least 200 mg / L, at least 250 mg / L, at least 300 mg / L, between 100 and 300 mg / L, between 100 and 500 mg / L, between 100 and 1000 mg / L, at least 1000 mg / L, at least 1250 mg/liter, at least 1500 mg/liter, at least about 1750 mg/liter, at least about 2000 mg/liter, at least about 10000 mg/liter, or more.
74. 74. The method of any one of the foregoing claims, wherein one or more subunits of said multi-subunit complex are expressed from more than one gene copy.
75. 75. The method of any one of the foregoing claims, wherein said multi-subunit complex comprises an antibody which is expressed from between 1-10 copies of a gene encoding the light chain of said antibody and from 1-10 copies of a gene encoding the heavy chain of said antibody.
76. 76. The method of any one of the foregoing claims, wherein genes that provide for expression of said multi-subunit complex are integrated into genome of said cells.
77. 77. The method of any one of claims 1-75, wherein genes that provide for expression of said multi-subunit complex are contained on an extrachromosomal element, plasmid, or artificial chromosome.
78. 78. The method of claim 76 or 77, wherein said cells comprise more copies of the gene that provide for the expression of the light chain of said antibody than copies of the gene that provide for expression of the heavy chain of said antibody.
79. 79. The method of claim 76 or 76, wherein the respective number of copies of the gene encoding the heavy chain of said antibody and the number of copies of the gene encoding the light chain of said antibody in said cells are: 2 and 2, 2 and 3, 3 and 3, 3 and 4, 3 and 5, 4 and 3, 4 and 4, 4 and 5, 4 and 6, 5 and 4, 5 and 5, 5 and 6, or 5 and 7.
80. 80. The method of claim 76 or 77, wherein the respective number of copies of the gene encoding the heavy chain of said antibody and the number of copies of the gene encoding the light chain of said antibody in said cells are: 2 and 1, 3 and 1, 4 and 1, 5 and 1, 6 and 1, 7 and 1, 8 and 1, 9 and 1, 10 and 1, 1 and 2, 2 and 2, 3 and 2, 4 and 2, 5 and 2, 6 and 2, 7 and 2, 8 and 2, 9 and 2, 10 and 2, 1 and 3, 2 and 3, 3 and 3, 4 and 3, 5 and 3, 6 and 3, 7 and 3, 8 and 3, 9 and 3, 10 and 3, 1 and 4, 2 and 4, 3 and 4, 4 and 4, 5 and 4, 6 and 4, 7 and 4, 8 and 4, 9 and 4, 10 and 4, 1 and 5, 2 and 5, 3 and 5, 4 and 5, 5 and 5, 6 and 5, 7 and 5, 8 and 5, 9 and 5, 10 and 5, 1 and 6, 2 and 6, 3 and 6, 4 and 6, 5 and 6, 6 and 6, 7 and 6, 8 and 6, 9 and 6, 10 and 6, 1 and 7, 2 and 7, 3 and 7, 4 and 7, 5 and 7, 6 and 7, 7 and 7, 8 and 7, 9 and 7, 10 and 7, 1 and 8, 2 and 8, 3 and 8, 4 and 8, 5 and 8, 6 and 8, 7 and 8, 8 and 8, 9 and 8, 10 and 8, 1 and 9, 2 and 9, 3 and 9, 4 and 9, 5 and 9, 6 and 9, 7 and 9, 8 and 9, 9 and 9, 10 and 9, 1 and 10, 2 and 10, 3 and 10, 4 and 10, 5 and 10, 6 and 10, 7 and 10, 8 and 10, 9 and 10, 10 and 10.
81. 81. The method of any one of the foregoing claims, wherein the culture of step (c) is grown in a production medium.
82. 82. The method of claim 81, wherein said production medium is a minimal medium.
83. 83. The method of claim 81, wherein said minimal medium lacks selective agents.
84. 84. The method of claim 81, wherein said minimal medium lacks pre-formed amino acids or other complex biomolecules.
85. 85. The method of claim 81, wherein the production medium is a complex medium.
86. 86. The method of claim 85, wherein the complex medium comprises one or more of yeast extract, soy peptones, and other plant peptones.
87. 87. The method of any one of the foregoing claims, wherein the culture of step (c) is grown to a high cell density.
88. 88. The method of claim 87, wherein said high cell density is at least 50 g/L.
89. 89. The method of claim 87, wherein said high cell density is at least 100 g/L.
90. 90. The method of claim 87, wherein said high cell density is at least 300 g/L.
91. 91. The method of claim 87, wherein said high cell density is at least 400 g/L.
92. 92. The method of claim 87, wherein said high cell density is at least 500 g/L.
93. 93. The method of claim 87, wherein said high cell density is at least 750 g/L.
94. 94. The method of any one of the foregoing claims, wherein the yeast cells are cultured for at least 20 doublings and maintain high levels of expression of said multi-subunit complex after said at least 20 doublings.
95. 95. The method of any one of the foregoing claims, wherein the cells of step (c) are cultured for at least 50 doublings and maintain high levels of expression of said multi-subunit complex after said at least 50 doublings.
96. 96. The method of any one of the foregoing claims, wherein the cells of step (c) are cultured for at least 100 doublings and maintain high levels of expression of said multi-subunit complex after said at least 100 doublings.
97. 97. The method of any one of the foregoing claims, wherein at least one subunit of said multi-subunit complex comprises a secretion signal.
98. 98. The method of claim 97, wherein said multi-subunit complex comprises an antibody.
99. 99. The method or composition of claim 97 or 98, wherein the secretion signal comprises one or more polypeptides selected from the group consisting of: SEQ ID NOS: 414 to 437 and any combination thereof.
100. 100. The method of any one of the foregoing claims, wherein said multi-subunit complex is not any of the antibodies disclosed in U.S. Provisional Application No. 61/418,832, filed December 1, 2010, PCT/US11/62963, filed December 1, 2011, U.S. Ser. No. 13/309,295, filed December 1, 2011, U.S. Ser. No. 13/309,153, filed December 1, 2011, U.S. Ser. No. 13/308,665 filed on December 1, 2011, and U.S. Ser. No. 13/308,831, filed December 1, 2011.
101. 101. The method of any one of the foregoing claims, wherein said multi-subunit complex is not Abl-NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, AblO-NGF, Abl 1-NGF, Abl2-NGF, Abl3-NGF, Abl4-NGF, AM5-NGF, Abl6-NGF, Abl7-NGF, Abl8-NGF, AM9-NGF, Ab20-NGF, and Ab21-NGF, or an Fab2 or Fabl fragment thereof.
102. 102. The method of any one of the foregoing claims, wherein said multi-subunit complex does not contain at least one, at least two, at least three, at least four, at least five, or at least all six of the complementarity determining regions (CDRs) contained in any of the following antibodies: Abl-NGF, Ab2-NGF, Ab3-NGF, Ab4-NGF, Ab5-NGF, Ab6-NGF, Ab7-NGF, Ab8-NGF, Ab9-NGF, AblO-NGF, Abl 1-NGF, Abl2-NGF, Abl3-NGF, Abl4-NGF, Abl5-NGF, Abl6-NGF, Abl7-NGF, AM8-NGF, Abl9-NGF, Ab20-NGF, or Ab21-NGF and optionally having binding specificity for NGF.
103. 103. The method of any one of the foregoing claims, wherein said multi-subunit complex does not comprise or consist of the light and heavy chain polypeptide sequences of SEQ ID NOs: 51 and 401, respectively, SEQ ID NOs: 53 and 402, respectively, SEQ ID NOs: 405 and 406, respectively, and SEQ ID NOs: 407 and 408, respectively.
104. 104. The method of any one of the foregoing claims, wherein said multi-subunit complex does not comprise an antibody containing at least one, at least two, at least three, at least four, at least five, or at least all six of the CDRs of SEQ ID NOs: 55, 56, 57, 58, 59, and 60, and optionally having binding specificity for NGF.
105. 105. The method of any one of the foregoing claims, wherein said multi-subunit complex does not comprise any of the antibodies or antibody coding sequences disclosed herein in the sections entitled “Anti-NGF Antibodies and Binding Fragments Thereof Having Binding Activity for NGF” and “Polynucleotides Encoding Anti-NGF Antibody Polypeptides.”