Anti-NGF antibodies and uses thereof

Abstract

The present invention provides novel anti-NGF proteins, antibodies, and NGF-binding fragments thereof that inhibit the association of NGF with TrkA and / or p75 and are suitable for administration to a canine or feline subject. The present invention also provides novel compositions and methods for treating pain or inducing an analgesic effect in a canine or feline subject, comprising administering an effective amount of an anti-NGF protein, antibody, or fragment thereof. The methods and compositions are used to treat or prevent NGF-related disorders.

Description

[Technical field]

[0001] Related Applications and Incorporation by Reference This application claims priority to U.S. Provisional Application No. 63 / 282,590, filed November 23, 2021, and U.S. Provisional Application No. 63 / 383,173, filed November 10, 2022, each of which is incorporated by reference in its entirety herein.

[0002] All documents cited or referenced herein ("documents cited herein"), and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, manuals, product specifications, and product brochures for the products referred to herein or in the documents incorporated by reference herein, are hereby incorporated by reference and may be employed in the practice of this invention. More specifically, all references are similarly incorporated by reference as if each individual document was expressly and individually indicated to be incorporated by reference.

[0003] Sequence Listing This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web, and is incorporated herein by reference in its entirety. The ASCII copy, created on November 22, 2022, is named G9432-99003.xml and is 292,984 bytes in size.

[0004] The present invention provides novel anti-NGF proteins, antibodies, and NGF-binding fragments thereof that inhibit the association of NGF with TrkA and / or p75 and are suitable for administration to a canine or feline subject. The present invention also provides novel compositions and methods for treating pain or inducing an analgesic effect in a canine or feline subject, comprising administering an effective amount of an anti-NGF protein, antibody, or fragment thereof. The methods and compositions are used to treat or prevent NGF-related disorders. [Background technology]

[0005] Nerve growth factor (NGF) is important for the development and maintenance of peripheral sympathetic neurons, fetal sensory neurons, and basal forebrain cholinergic neurons. NGF upregulates the expression of neuropeptides in sensory neurons, and its activity is mediated through two distinct membrane-bound receptors. Several neurotropins (NTs), including NGF, bind to a low-affinity receptor identified as p75. NGF selectively binds to the high-affinity neurotrophin receptor TrkA, with high affinity.

[0006] Upon neurotrophin binding, TrkA not only undergoes autophosphorylation but also phosphorylates members of the MAPK pathway, the presence of which may play a role in directing cellular differentiation and specifying sensory neuron subtypes.

[0007] NGF plays a role in several diseases and disorders, including but not limited to the pain associated with a wide range of diseases and disorders, such as cancer-related pain, neuropathic pain, and neurogenic pain.Due to the involvement of NGF in a wide range of pain-related diseases and disorders, there is a need in the art for compositions and methods useful for preventing or treating NGF-related diseases and disorders, particularly pain-related diseases and disorders, including dogs, cats, and other animals.Particularly preferred anti-NGF compositions are those that cause minimal or minimal adverse reactions, such as inflammation, when administered to a subject.

[0008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0009] The present invention provides novel anti-NGF binding proteins for the treatment or amelioration of NGF-related disorders and is particularly suitable for use in, but not limited to, dogs and cats.

[0010] The present invention provides binding proteins that specifically bind to NGF. In certain embodiments, the binding proteins are optimized for administration to dogs. In certain embodiments, the binding proteins are optimized for administration to cats.

[0011] In one aspect, the present invention provides binding proteins designed or adapted to bind NGF in the manner of an antibody, i.e., by one or more complementarity determining regions (CDRs). CDRs can be identified by the International ImMunoGeneTics (IMGT) information system. Thus, in a particular embodiment, the anti-NGF binding protein comprises: (a) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 146), where X1 comprises A, G, or N, X2 comprises L or M, X3 comprises A, D, E, or S, X4 comprises F, I, L, M, or V, X5 comprises N or T, and X6 comprises CX3, CX4, CX5, CX6, CX7, CX8, CX9, CX10, CX11, CX12, CX13, CX14, CX15, CX16, CX17, CX18, CX19, CX20, CX21, CX22, CX23, CX24, CX25, CX26, CX27, CX28, CX30, CX31, CX32, CX33, CX34, CX35, CX36, CX37, CX38, CX39, CX40, CX41, CX42, CX43, CX44, CX45, CX46, CX47, CX48, CX49, CX48, CX49, CX41, CX42, CX43, CX44, CX45, CX45, CX46, CX47, CX48, CX49, CX49, CX49, CX49, CX49, CX49, CX49, CX49, CX49 X comprises E, S, or T, X7 comprises G, H, N, S, or Q, and X comprises A or S; (b) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence X1X2SNX5GT (SEQ ID NO: 147), where X comprises I or L, X2 comprises W or Y, and X5 comprises G or R; (c) an amino acid sequence AX2IX4X5YX7X8X9YLX 12 X 13 YX 15 X 16 X 17 (SEQ ID NO: 148), wherein X2 comprises D, E, K, N, Q, S, or T; X4 comprises W or Y; X5 comprises F, H, W, or Y; X7 comprises D or E; X8 comprises A or S; X9 comprises D or Y; and X 12 contains H or Y, and X 13 contains F or W, and X 15 contains F, I, L, W, or Y, and X 16 contains D or Q, and X 17comprises F, I, L, M, W, or Y), (d) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence X1X2IX4X5X6 (SEQ ID NO: 149), wherein X1 comprises D, E, or K, X2 comprises A, G, or N, X4 comprises G, N, Q, or S, X5 comprises N or S, and X6 comprises A, G, N, S, or T), (e) a light chain complementarity determining region 2 (VL-CDR3) comprising the amino acid sequence AX2X3 (SEQ ID NO: 150), and (f) a light chain complementarity determining region 3 (VL-CDR2) comprising the amino acid sequence QX2GX4X5X6PX8T (SEQ ID NO: 151), where X2 comprises H or Q, X4 comprises F, H, W or Y, X5 comprises K or Q, X6 comprises F or W, and X8 comprises L or M.

[0012] In certain embodiments, the anti-NGF binding protein comprises: (a) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 152), where X1 comprises A or G, X2 comprises L or M, X3 comprises E or S, X4 comprises F or L, X5 comprises N or T, X6 comprises E, S, or T, X7 comprises H, N, or S, and X8 comprises A or S; (b) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence X1WSNX5GT (SEQ ID NO: 153), where X1 comprises I or L, and X5 comprises G or R; (c) an amino acid sequence of AX2IYYYX7ADYLHX 13 YX 15 DX 17 (SEQ ID NO: 154) (wherein X2 comprises N, Q, S, or T; X7 comprises D or E; and 13 contains F or W, and X 15 contains F, I, L, W, or Y, and X 17X4 comprises F, I, L, or M; (d) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence X1GIX4X5X6 (SEQ ID NO: 155), where X1 comprises D or E, X4 comprises N, Q, or S, X5 comprises N or S, and X6 comprises G, N, S, or T; (e) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence ATX3 (SEQ ID NO: 156), where X3 comprises D, E, N, Q, or S; and (f) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence QQGX4X5X6PX8T (SEQ ID NO: 157), where X4 comprises F, H, W, or Y, X5 comprises K or Q, X6 comprises F or W, and X8 comprises L or M.

[0013] In certain embodiments, the anti-NGF binding protein comprises a heavy chain CDR1 as shown in Figure 1. In certain embodiments, the anti-NGF binding protein comprises a heavy chain CDR2 as shown in Figure 1. In certain embodiments, the anti-NGF binding protein comprises a heavy chain CDR3 as shown in Figure 1. In certain embodiments, the anti-NGF binding protein comprises a light chain CDR1 as shown in Figure 2. In certain embodiments, the anti-NGF binding protein comprises a light chain CDR2 as shown in Figure 2. In certain embodiments, the anti-NGF binding protein comprises a light chain CDR3 as shown in Figure 2.

[0014] In certain embodiments, the anti-NGF binding protein comprises the heavy chain CDRs of the heavy chain variable domain depicted in FIG.

[0015] In certain embodiments, the anti-NGF binding protein is V H a heavy chain variable domain (V H ).

[0016] In certain embodiments, the anti-NGF binding protein comprises the light chain CDRs of the light chain variable domain depicted in FIG.

[0017] In certain embodiments, the anti-NGF binding protein comprises a light chain variable domain (V) that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or identical to the light chain variable domains shown in FIG. L ).

[0018] In certain embodiments, the anti-NGF binding protein comprises a VFv derived from the Fvs shown in FIG. H and V L Includes.

[0019] In Figures 1 and 2, the CDRs are identified according to the IMGT system. Alternatively, the CDRs may be identified according to the Kabat or Chothia numbering systems. Thus, in certain embodiments, the anti-NGF binding protein comprises an antigen-binding portion that comprises one or more of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 according to the Kabat or Chothia numbering systems as further described herein.

[0020] V as described herein H Domain and V L In domain pairing, any V H Domain can be any V L can be used with any V H The CDR of the domain can be any V L In one embodiment, the antibody of the invention can be used with the CDRs of the V domain of SEQ ID NO: 137. H CDR (SC-42_101) and V of SEQ ID NO:3 L In an embodiment comprising the amino acid arginine at position 55 of VH-CDR2, the antibody of the invention comprises the VH-CDR of SEQ ID NO: 207. H CDR (SC-42_101R) and V of SEQ ID NO:3 L Includes CDR(SC-42_006).

[0021] In certain embodiments, the antibodies of the invention include V H and V L Domains are built in. V selected together H and V L Clones are identified in both Figure 1 and Figure 2 as having the same clone name. Similarly, V identified by the clone name in Figure 1 H The CDRs of the domains are identified by the same clone names in Figure 2. L can be used with the CDRs of the domain. H and V L Domains may include conservative substitutions, such as, but not limited to, V H CDR and V L Specific positions of CDRs, V H and V L The positions adjacent to the CDRs, as well as the V H Domain and V L It may further include conservative mutations observed at domain positions.

[0022] In certain embodiments, an antibody of the invention comprises the VLK of clone 2166, SC-42_006, SC-42_007, SC-42_008, SC-42_010, SC-42_011, SC-42_023, SC-42_032, SC-42_045, SC-42_047, SC-42_048, SC-42_052, SC-42_070, SC-42_073, SC-42_077, SC-42_082, SC-42_090, or SC-42_101. H and V L Contains the CDRs (Figures 1 and 2).

[0023] In certain embodiments, an antibody of the invention has a V sequence that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or identical to clone 2166, SC-42_006, SC-42_007, SC-42_008, SC-42_010, SC-42_011, SC-42_023, SC-42_032, SC-42_045, SC-42_047, SC-42_048, SC-42_052, SC-42_070, SC-42_073, SC-42_077, SC-42_082, SC-42_090, or SC-42_101. H and V L Includes the domain.

[0024] In certain embodiments, the fetinized anti-NGF binding protein has the amino acid sequence: (a) X1LX3X4X5X6X7X8MX 10 (SEQ ID NO:208), wherein X1 comprises A, G, L, N, or Q; X3 comprises A, D, E, G, H, I, M, S, T, or Y; X4 comprises L, M, or V; X5 comprises A, M, N, R, S, T, or V; X6 comprises A, E, G, H, K, R, S, or T; X7 comprises A, D, H, I, N, Q, S, T, or Y; X8 comprises A or S; and X9 comprises A or S. 10 contains S or V), (b) the amino acid sequence X1X2X3X4X5GTX8YX 10 DX 12 VX 14 (SEQ ID NO: 209), wherein X1 comprises I or L, X2 comprises W or Y, X3 comprises A, P, or S, X4 comprises D, E, N, Q, R, or S, X5 comprises G, R, or Y, X8 comprises D or Y, and X 10 contains D, E, H, S, or T, and X 12 contains D or S, and X 14 contains D, E, or K), (c) the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 LX 12 X 13 X 14 FX16 X 17 (SEQ ID NO:210), wherein X1 comprises A, D, E, K, N, Q, S, or T; X2 comprises A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X3 comprises I, L, W, or Y; X4 comprises F, T, W, or Y; X5 comprises F, H, or Y; X6 comprises H or Y; X7 comprises D or E; X8 comprises A, S, or V; X9 comprises D, E, H, K, N, Q, or Y; and X 10 contains F, H, or Y, and X 12 contains H or Y, and X 13 contains F or W, and X 14 contains D, I, L, W, or Y, and X 16 contains D or Q, and X 17 contains E, F, H, I, L, M, N, P, W, or Y), (d) the amino acid sequence X1ASX4X5X6X7X8X9LX 11 (SEQ ID NO:211), wherein X1 comprises F or R, X4 comprises E, K, or N, X5 comprises A or G, X6 comprises I, L, or V, X7 comprises A, D, G, L, P, Q, S, V, or Y, X8 comprises K, Q, N, S, or Y, and X9 comprises A, D, E, F, G, H, K, L, N, Q, R, S, or T; and X 11X2 comprises A, G, or S; (e) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence AX2X3X4X5X6X7 (SEQ ID NO: 212), or T), and (f) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence QQX3X4X5X6X7X8T (SEQ ID NO: 213), where X3 comprises G or Y; X4 comprises D, F, G, H, K, L, R, S, T, V, W, or Y; X5 comprises E, K, Q, R, or S; X6 comprises I, F, T, or W; X7 comprises E or P; and X8 comprises L, M, or W.

[0025] In certain embodiments, the anti-NGF binding protein comprises a heavy chain CDR1 as shown in Figure 17A. In certain embodiments, the anti-NGF binding protein comprises a heavy chain CDR2 as shown in Figure 17A. In certain embodiments, the anti-NGF binding protein comprises a heavy chain CDR3 as shown in Figure 17A. In certain embodiments, the anti-NGF binding protein comprises a light chain CDR1 as shown in Figure 17B. In certain embodiments, the anti-NGF binding protein comprises a light chain CDR2 as shown in Figure 17B. In certain embodiments, the anti-NGF binding protein comprises a light chain CDR3 as shown in Figure 17B.

[0026] In certain embodiments, the anti-NGF binding protein comprises the heavy chain CDRs of the heavy chain variable domain depicted in Figure 17A.

[0027] In certain embodiments, the anti-NGF binding protein is V H A heavy chain variable domain (V H ).

[0028] In certain embodiments, the anti-NGF binding protein comprises the light chain CDRs of the light chain variable domain depicted in Figure 17B.

[0029] In certain embodiments, the anti-NGF binding protein comprises a light chain variable domain (V) that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or identical to the light chain variable domain shown in FIG. 17B. L ).

[0030] V shown in FIG. H The CDRs are the V L The V and CDRs can be used in any combination. H The domain is V shown in FIG. L Any combination with the domains can be used. Tables 11 and 12 show example combinations.

[0031] In certain embodiments, the anti-NGF binding protein comprises a V from an Fv. H and V L Including V H is shown in FIG. 17A, and V L is shown in FIG. 17B.

[0032] In Figure 17, the CDRs are identified by the IMGT system. Alternatively, the CDRs may be identified according to the Kabat or Chothia numbering systems. Thus, in certain embodiments, the anti-NGF binding protein comprises an antigen-binding portion that comprises one or more of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 according to the Kabat or Chothia or IMGT numbering systems as further described herein.

[0033] In certain embodiments, the anti-NGF binding protein comprises CDRs as described above, with the further restriction that each CDR comprises no more than one or two amino acid differences compared to the specific antibody heavy and light chains described herein, e.g., the heavy and light chain CDRs whose sequences are shown in Figures 1, 2, 17A, and 17B (which are of similar sequence and bind NGF with high affinity). H and V L Contains the CDRs of.

[0034] In certain embodiments, the anti-NGF protein contains no more than one or two amino acid differences per CDR compared to the heavy and light chains of certain caninized antibodies described herein, e.g., the heavy and light chain CDRs described in Figures 1 and 2, which are of similar sequence and bind NGF with the highest affinity. Such antibodies include those which contain no more than two changes per VH-CDR, i.e. two, one or no changes per CDR compared to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137 or SEQ ID NO:141, and no more than two changes per VL-CDR, i.e. two, one or no changes per CDR compared to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138 or SEQ ID NO:142.

[0035] In certain embodiments, the anti-NGF protein contains no more than one or two amino acid differences per CDR compared to the specific fetinized antibody heavy and light chains described herein, e.g., the heavy and light chain CDRs described in Figures 17A and 17B, which are of similar sequence and bind NGF with the highest affinity. Such antibodies include those which contain no more than two changes per VH-CDR, i.e., two, one or no changes per CDR compared to SEQ ID NO:141, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, or SEQ ID NO:206, and no more than two changes per VL-CDR, i.e., two, one or no changes per CDR compared to SEQ ID NO:142, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, or SEQ ID NO:203.

[0036] Mutations and combinations thereof within and between CDRs, including tolerated and favored mutations, are evident from the sequence data sets shown in Figures 1 and 2, and Figures 17A and 17B. For example, by comparing sequence diversity or lack thereof at various CDR positions across the data set, CDR positions where particular amino acids are favored for binding can be observed. Similarly, V H Interchain or V L By comparing sequence diversity between chains, it is possible to observe CDR positions where amino acid changes may be cooperative. Furthermore, this data set allows the identification of CDR positions that are likely to be important for binding.

[0037] Certain antibodies disclosed herein were selected from dog or cat libraries based on CDR sequence similarity with other anti-NGF antibodies. Thus, both CDRs and FRs are dog-like or cat-like, and a certain degree of homogeneity will be observed between antibody heavy and light chains derived from the same germline sequence. It is understood that such homogeneity is not required and is a result of the caninization and felineization methods employed. It is also understood that sequence variation can be tolerated or introduced into the FRs to the extent that it is not detrimental to antigen binding. In certain embodiments, the anti-NGF protein comprises a heavy chain framework (FR1H+FR2H+FR3HH) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to the heavy chain shown in FIG. 1. In certain embodiments, the anti-NGF protein comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, or SEQ ID NO:141.

[0038] In certain embodiments, the anti-NGF protein comprises a heavy chain framework (FR1H+FR2H+FR3HH) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to the heavy chain depicted in Figure 17 A. In certain embodiments, the anti-NGF protein comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:141, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, or SEQ ID NO:206.

[0039] In certain embodiments, the anti-NGF protein comprises a light chain framework (FR1+FR2+FR3+FR4) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to the light chain depicted in Figure 2. In certain embodiments, the anti-NGF protein comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:142.

[0040] In certain embodiments, the anti-NGF protein comprises a light chain framework (FR1+FR2+FR3+FR4) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to the light chain depicted in Figure 17B. In certain embodiments, the anti-NGF protein comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:142, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, or SEQ ID NO:203.

[0041] For a discussion of the naturally conserved networks of amino acids that support antibody V(H) and V(L) function, see, e.g., Wang et al., Conserved amino acid networks involved in antibody variable domain interactions. Proteins 2009 Jul;76(1):99-114. Wang describes the V(H) and V(L) functions of antibodies. H and V L Domain, V H -C H 1 Variable-constant domain interface, as well as V L and C H Camelidae V evolved to lack interaction with 1 HHConserved and non-conserved amino acid pairs in the domains were identified. In certain embodiments, mutations are introduced to optimize the biopharmaceutical and biophysical properties, such as efficacy, safety, and manufacturability, as well as stability, of therapeutic antibodies. See, e.g., Douillard et al., Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics. Antibodies (Basel) 2019 Sep 6; 8(3): 46.

[0042] In certain embodiments, the invention provides an isolated recombinant NGF binding protein, wherein the variable heavy chain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the heavy chain variable domain depicted in Figure 1. In certain embodiments, the heavy chain variable domain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, or SEQ ID NO:137.

[0043] In certain embodiments, the invention provides an isolated recombinant NGF binding protein, wherein the variable light chain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the light chain variable domain depicted in Figure 2. In certain embodiments, the light chain variable domain comprises an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, or SEQ ID NO:138.

[0044] In another aspect, the present invention provides a nucleic acid encoding the anti-NGF protein of the present invention.In another aspect, the present invention provides a vector comprising a nucleic acid encoding the anti-NGF protein of the present invention.

[0045] In another aspect, the invention provides a cell comprising a nucleic acid of a vector of the invention or expressing an anti-NGF protein of the invention.

[0046] Anti-NGF binding proteins, including but not limited to antibodies and antibody fragments, specifically bind NGF and inhibit the association of NGF with TrkA and further inhibit the association of NGF with p75. In certain embodiments, these novel anti-NGF binding proteins are suitable for detecting NGF and treating pain and pain-related disorders and conditions, such as inflammation-related pain, cancer, certain pain and inflammation-related disorders, particularly pain-related disorders associated with elevated NGF levels, and may be administered alone or in combination with another active agent, such as another biologic, including but not limited to an NSAID or an opioid analgesic.

[0047] Therefore, it is the object of the present invention not to include within the present invention any previously known products, processes for making products, or methods for using products, so as to disclose any previously known products, processes, or methods to which the applicant has reserved rights. It is further noted that the present invention is not intended to include within the scope of the present invention any products, processes, or methods for making products, or methods for using products, that do not meet the written description and enablement requirements of the USPTO (Article 112, first paragraph) or the EPO (Article 83 EPC), so as to disclose any previously known products, processes for making products, or methods for using products, to which the applicant has reserved rights. In the practice of the present invention, it may be advantageous to comply with Article 53(c) of the EPC and Rules 28(b) and (c) of the EPC. All rights are expressly reserved to explicitly exclude any embodiment that is the subject of any granted patent(s) of the applicant in the line of this application or in any other line, or in any prior application of any third party. Nothing herein should be construed as a promise.

[0048] It should be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like can have the meaning ascribed to them in U.S. patent law, e.g., they can mean "include," "included," "including," and the like, and terms such as "consisting essentially of" and "consists essentially of" can have the meaning ascribed to them in U.S. patent law, e.g., they allow for elements not expressly recited, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention.

[0049] These and other embodiments are disclosed or are obvious from and are encompassed by the following detailed description. [Brief description of the drawings]

[0050] [Figure 1A] 1 shows an alignment of the amino acid sequences of exemplary VH heavy chain variable domains of the present invention. The aligned variable domains are divided into two parts: part (A) shows the N-terminus. For the purposes of alignment, the framework (FR) and complementarity determining regions (CDR) are identified according to the IMGT system. The CDRs and FRs may also be mapped according to other systems disclosed herein. [Figure 1B] Figure 1 shows an alignment of amino acid sequences of exemplary VH heavy chain variable domains of the invention. The aligned variable domains are divided into two parts: part (B) shows the C-terminus. For the purpose of alignment, framework (FR) and complementarity determining regions (CDR) are identified according to the IMGT system. CDRs and FRs may also be mapped according to other systems disclosed herein. [Figure 2A] Figure 1 shows an alignment of the amino acid sequences of exemplary Vκ light chain variable domains of the invention. The aligned variable domains are divided into two parts: part (A) shows the N-terminus. For alignment purposes, the framework (FR) and complementarity determining regions (CDR) are identified according to the IMGT system. The CDRs and FRs may also be mapped according to other systems disclosed herein. Each VL domain is suitable for pairing with any of the VH domains shown in Figure 1. In conjunction with Figure 1, the names of the clones indicate a selection of exemplary VH-VL pairs to be tested for binding. [Figure 2B]Figure 1 shows an alignment of the amino acid sequences of exemplary Vκ light chain variable domains of the invention. The aligned variable domains are divided into two parts: part (B) shows the C-terminus. For alignment purposes, the framework (FR) and complementarity determining regions (CDR) are identified according to the IMGT system. The CDRs and FRs may also be mapped according to other systems disclosed herein. Each VL domain is suitable for pairing with any of the VH domains shown in Figure 1. In conjunction with Figure 1, the names of the clones indicate a selection of exemplary VH-VL pairs to be tested for binding. [Diagram 3] Shows inhibition of proliferation of TF-1 cells. [Figure 4] The amino acid sequences of the heavy (SEQ ID NO: 144) and light (SEQ ID NO: 145) chains of the chimeric 2166 antibody are shown. Two residue changes ("AA", underlined and bold) were made in the Fc to eliminate effector activity. This change is similar to the "LALA" mutation described for human IgG1 Fc. The chimeric 2166 antibody contains a canine IgGB heavy chain constant region and a kappa light chain constant region. [Diagram 5] 16 is a sensorgram of canine 2166 chimeric antibody binding to canine NGF. NGF concentrations were 0.78, 1.56, 3.12, 6.25, and 12.5 nM. [Figure 6] Sensorgrams of canine NGF alone binding to canine p75-Fc. NGF concentrations were 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM. [Figure 7] Sensorgrams of canine NGF alone binding to canine TrkA-Fc. NGF concentrations were 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM. [Figure 8] Sensorgram of canine 2166 chimeric antibody-NGF mixture binding to canine p75-Fc. NGF concentrations were 12.5, 25, and 50 nM. [Figure 9] Sensorgram of canine 2166 chimeric antibody-NGF mixture binding to canine TrkA-Fc. NGF concentrations were 12.5, 25, and 50 nM. [Figure 10] Sensorgrams of 70 caninized clones binding to canine NGF are shown. NGF concentrations were 0.23, 0.69, 2.06, 6.17, and 18.52 nM. [Figure 11] Sensorgrams of canine NGF alone binding to canine p75-Fc. NGF concentrations were 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM. [Figure 12] Sensorgrams of canine NGF alone binding to canine TrkA-Fc. NGF concentrations were 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM. [Figure 13] 1 shows a sensorgram of the caninized SC42_101 antibody-NGF mixture binding to canine p75-Fc. NGF concentrations were 12.5, 25, and 50 nM. [Figure 14] 1 shows a sensorgram of the caninized SC42_101 antibody-NGF mixture binding to canine TrkA-Fc. NGF concentrations were 12.5, 25, and 50 nM. [Figure 15] The VH (sequence number 141) and VL (sequence number 142) amino acid sequences of a fetinized anti-NGF antibody are shown. [Figure 16] Sensorgram of feline clone 101 binding to NGF. NGF concentrations were 1.23, 3.7, 11, 33, and 100 nM. [Figure 17A] 1 shows an alignment of the amino acid sequences of exemplary felineized and affinity matured felineized VH heavy chain variable domains of the present invention. For purposes of alignment, frameworks (FRs) and complementarity determining regions (CDRs) are identified according to the IMGT system. CDRs and FRs may also be mapped according to other systems disclosed herein. See, for example, Table 4 and CDRs defined using a combination of the Kabat and IMGT approaches. [Figure 17B]1 shows an alignment of the amino acid sequences of exemplary felineized and affinity matured felineized Vκ light chain variable domains of the present invention. For purposes of alignment, frameworks (FRs) and complementarity determining regions (CDRs) are identified according to the IMGT system. CDRs and FRs may also be mapped according to other systems disclosed herein. See, for example, Table 4 and CDRs defined using a combination of the Kabat and IMGT approaches. [Figure 18A] Shown is a sensorgram of affinity matured feline antibody: clone 101. NGF concentrations were 50, 25, 12.5, 6.25, 3.125, and 1.56 nM. [Figure 18B] Shown is a sensorgram of affinity matured feline antibody: AHF17602. NGF concentrations were 50, 25, 12.5, 6.25, 3.125, and 1.56 nM. [Figure 18C] Shown is a sensorgram of affinity matured feline antibody: SC-184_76. NGF concentrations were 50, 25, 12.5, 6.25, 3.125, and 1.56 nM. [Figure 18D] Shown is a sensorgram of affinity matured feline antibody: SC-184_76-Arg. NGF concentrations were 50, 25, 12.5, 6.25, 3.125, and 1.56 nM. [Figure 18E] Shown is a sensorgram of affinity matured feline antibody: SC-102. NGF concentrations were 50, 25, 12.5, 6.25, 3.125, and 1.56 nM. [Figure 18F] Shown is a sensorgram of affinity matured feline antibody: SC-184_102-Arg. NGF concentrations were 50, 25, 12.5, 6.25, 3.125, and 1.56 nM. [Figure 18G] Shown is a sensorgram of affinity matured feline antibody: SC-110. NGF concentrations were 50, 25, 12.5, 6.25, 3.125, and 1.56 nM. [Figure 18H] Shown is a sensorgram of affinity matured feline antibody: SC-184_110-Arg. NGF concentrations were 50, 25, 12.5, 6.25, 3.125, and 1.56 nM.

[0051] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0052] The following detailed description, given by way of example and not intended to limit the invention to only the specific embodiments described, can be best understood in conjunction with the accompanying drawings, in which: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] According to certain exemplary embodiments of the present invention, the NGF binding protein is an anti-NGF antibody or an antigen-binding fragment thereof. As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (referred to herein as HCVR or V H The heavy chain constant region is made up of three domains, C H 1. C H 2, and C H Each light chain comprises a light chain variable region (herein referred to as LCVR or V L The light chain constant region comprises one domain (C L ) is included. V H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V Lconsists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-NGF antibody (or antigen-binding portion thereof) may be identical to the canine germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs.

[0054] The antibody residues that have a substantial effect on the affinity and specificity of binding to the target antigen are mainly present in the CDRs. Kabat et al. first proposed a standardized numbering scheme for the variable regions of immunoglobulins by compiling and aligning the sequences of heavy and light immunoglobulin chains and identifying conserved and hypervariable regions and residues. (Kabat EA et al., 1979, Sequences of Immunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain and BP-Microglobulins, Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health). The Kabat system is a widely adopted standard for numbering antibody residues, but the hypervariable regions defined by Kabat do not exactly match the structural features of the antigen-binding loops. Chothia and Lesk developed a structure-based numbering scheme by aligning crystal structures of antibody variable regions and classified the CDR loops into a few "canonical" classes (Chothia C, et al., 1987, Canonical structure for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196:901-17. doi:10.1016 / 0022-2836(87)90412-8). The advantage of the Chothia numbering scheme is that topologically aligned residues of different antibodies are localized to the same position numbers, and Chothia's CDR definitions correspond to the structural antigen-binding loops in most antibody sequences.Lefranc introduced a new system based on germline sequences that aimed to standardize the numbering of all proteins of the immunoglobulin superfamily, including the T cell receptor chains (Giudicelli V et al., 1997, IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 25:206-11), which was then extended to the entire variable domain (Lefranc MP et al., 2003, IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. 27:55-77. doi:10.1016 / S0145-305X(02)00039-3). Non-conventional frameworks are aligned (Abhinandan KR et al., 2008, Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. 45:3832-9. doi:10.1016 / j.molimm.2008.05.022) and the variable chain sequence is subdivided into multiple fragments containing a structurally invariant "core" (Gelfand et al., 1998, Algorithmic determination of core positions in the V. L and V HAdditional numbering systems have been proposed for the identification of CDR residues in the domains of immunoglobulin molecules. J Comput Biol. (1998) 5:467-77. In certain embodiments of the invention, the CDR residues are identified according to the standard system as described above. In certain embodiments, the antibodies of the invention are identified by all or a subset of the Kabat CDR residues of the antibody sequences described herein. In certain embodiments, the antibodies of the invention are identified by all or a subset of the Chothia CDR residues of the antibody sequences described herein. In certain embodiments, the antibodies of the invention are identified by all or a subset of the IMGT CDR residues of the antibody sequences described herein. In certain embodiments, the antibodies of the invention are identified by CDR residues defined by two or more systems, including, for example and without limitation, all or a subset of the residues of VH-CDR1 according to Kabat, all or a subset of the residues of VH-CDR2 according to Chothia, all or a subset of the residues of VH-CDR3 according to Kabat, all or a subset of the residues of VL-CDR1 according to Kabat, all or a subset of the residues of VL-CDR2 according to IMGT, and all or a subset of the residues of VL-CDR3 according to Chothia. Table 1 shows the correspondence of FRs and CDRs for the antibody sequences shown in Figures 1 and 2. [Table 1]

[0055] Upon identification of the CDRs by Kabat, in certain embodiments, the caninized anti-NGF binding protein comprises: (a) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence X1X2X3X4X5 (SEQ ID NO: 158), where X1 comprises E, S, or T, X2 comprises G, H, N, S, or Q, X3 comprises A or S, X4 comprises I, M, or V, and X5 comprises D or S; (b) a heavy chain complementarity determining region 2 (VH-CDR3) comprising the amino acid sequence X1X2X3SNX6GTX9YX 11 X 12 AX 14 X 15 X 16(SEQ ID NO: 159), wherein X1 comprises V, L, M, or T; X2 comprises I or L; X3 comprises W or Y; X6 comprises G or R; X9 comprises D, Q, or S; and X 11 contains A, N, or T, and X 12 contains D or S, and X 14 contains I or V, and X 15 contains E or K, and X 16 contains G or S), (c) the amino acid sequence IX2X3YX5X6X7YLX 10 X 11 YX 13 X 14 X 15 (SEQ ID NO: 160), wherein X2 comprises W or Y, X3 comprises F, H, W, or Y, X5 comprises D or E, X6 comprises A or S, X7 comprises D or Y, and X 10 contains H or Y, and X 11 contains F or W, and X 13 contains F, I, L, W, or Y, and X 14 contains D or Q, and X 15 contains F, I, L, M, W, or Y), (d) the amino acid sequence X1ASX4X5IX7X8X9X 10 X 11 (SEQ ID NO: 161), wherein X1 comprises L or R, X4 comprises D, E, or K, X5 comprises A, G, or N, X7 comprises G, N, Q, or S, X8 comprises N or S, and X9 comprises A, G, N, S, or T; 10 contains L or V, and X 11X2 comprises A or N; (e) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence AX2X3X4X5X6X7 (SEQ ID NO: 162), wherein X2 comprises A, S, or T; X3 comprises A, D, E, N, Q, S, or T; X4 comprises A, E, K, L, N, Q, S, or T; X5 comprises L, M, or N; X6 comprises A or Q; and X7 comprises G, D, R, S, or T. and (f) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence X1X2GX4X5X6PX8T (SEQ ID NO: 163), where X1 comprises H, M, Q, or R; X2 comprises H, N, Q, or S; X4 comprises F, H, W, or Y; X5 comprises K or Q; X6 comprises F or W; and X8 comprises L or M.

[0056] In certain embodiments, the caninized anti-NGF binding protein comprises: (a) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence X1X2X3X4S (SEQ ID NO: 164), where X1 comprises E, S, or T, X2 comprises H or N, X3 comprises A or S, and X4 comprises I or M; (b) an amino acid sequence of TIWSNX6GTDYX 11 X 12 AVKG (SEQ ID NO: 165) (wherein X6 contains G or R; 11 contains A or T, and X 12 contains D or S), (c) the amino acid sequence IYYYX5ADYLX 10 X 11 YX 13 DX 15 (SEQ ID NO: 166) (wherein X5 comprises D or E; 10 contains H or Y, and X 11 contains F or W, and X 13 contains F, I, L, W, or Y, and X 15 contains F, I, L, or M), (d) the amino acid sequence RASEGIX7X8X9X 10X7 comprises N, Q, or S; X8 comprises N or S; X9 comprises G, N, S, or T; and X 10 X3 comprises L or V), (e) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence ATX3X4LX6X7 (SEQ ID NO: 168), where X3 comprises A, D, E, N, Q, or S, X4 comprises E, K, Q, or S, X6 comprises A or Q, and X7 comprises R or T, and (f) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence QQGX4X5X6PLT (SEQ ID NO: 169), where X4 comprises F, H, W, or Y, X5 comprises K or Q, and X6 comprises F or W.

[0057] According to the CDR identification by Chothia, in certain embodiments, the anti-NGF binding protein comprises: (a) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 170), where X1 comprises A, G, or N, X2 comprises L or M, X3 comprises A, D, E, or S, X4 comprises F, I, L, M, or V, X5 comprises N or T, X6 comprises E, S, or T, and X7 comprises G, H, N, S, or Q; (b) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence X1SNX4G (SEQ ID NO: 171), where X1 comprises W or Y, and X4 comprises G or R; (c) a heavy chain complementarity determining region 3 (VH-CDR4) comprising the amino acid sequence IX2X3YX5X6X7YLX 10 X 11 YX 13 X 14 X 15 (SEQ ID NO: 172), wherein X2 comprises W or Y, X3 comprises F, H, W, or Y, X5 comprises D or E, X6 comprises A or S, X7 comprises D or Y, and X 10 contains H or Y, and X 11 contains F or W, and X 13 contains F, I, L, W, or Y, and X 14 contains D or Q, and X 15comprises F, I, L, M, W, or Y); (d) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence SX2X3IX5X6X7 (SEQ ID NO: 173), wherein X2 comprises D, E, or K, X3 comprises A, G, or N, X5 comprises G, N, Q, or S, X6 comprises N or S, and X7 comprises A, G, N, S, or T; (e) a light chain complementarity determining region 2 (VL-CDR3) comprising the amino acid sequence AX2X3 (SEQ ID NO: 174), wherein X2 comprises D, E, or K, X3 comprises A, G, or N, X5 comprises G, N, Q, or S, X6 comprises N or S, and X7 comprises A, G, N, S, or T; and (f) a light chain complementarity determining region 2 (VL-CDR2), where X2 comprises A, S, or T, and X3 comprises A, D, E, N, Q, S, or T, and an antigen-binding portion comprising one or more of the following: (a) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence GX2X3X4PX6 (SEQ ID NO: 175), where X2 comprises F, H, W, or Y, X3 comprises K or Q, X4 comprises F or W, and X6 comprises L or M.

[0058] In certain embodiments, the anti-NGF binding protein comprises: (a) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 176), where X1 comprises A, G, or N, X2 comprises L or M, X3 comprises A, E, or S, X4 comprises F or L, X5 comprises N or T, X6 comprises E, S, or T, and X7 comprises H, N, or S; (b) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence WSNX4G (SEQ ID NO: 177), where X4 comprises G or R; (c) an amino acid sequence of IYX3YX5ADYLX 10 X 11 YX 13 DX 15 (SEQ ID NO: 178) (wherein X3 comprises F or Y, X5 comprises D or E, and X 10 contains H or Y, and X 11 contains F or W, and X 13 contains F, I, L, W, or Y, and X 15X2 comprises F, I, L, M, W, or Y), (d) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence SX2GIX5X6X7 (SEQ ID NO: 179), where X2 comprises D or E, X5 comprises N, Q, or S, X6 comprises N or S, and X7 comprises G, N, S, or T), (e) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence ATX3 (SEQ ID NO: 180), where X3 comprises D, E, N, Q, or S, and (f) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence GX2X3X4PX6 (SEQ ID NO: 181), where X2 comprises F, H, W, or Y, X3 comprises K or Q, X4 comprises F or W, and X6 comprises L or M.

[0059] In another aspect, the invention provides binding proteins suitable for use in mammals, such as, but not limited to, cats. In certain embodiments, the felineized anti-NGF binding protein has the amino acid sequence: (a) X1LX3X4X5X6X7X8MX 10 (SEQ ID NO:208), wherein X1 comprises A, G, L, N, or Q; X3 comprises A, D, E, G, H, I, M, S, T, or Y; X4 comprises L, M, or V; X5 comprises A, M, N, R, S, T, or V; X6 comprises A, E, G, H, K, R, S, or T; X7 comprises A, D, H, I, N, Q, S, T, or Y; X8 comprises A or S; and X9 comprises A or S. 10 contains S or V), (b) the amino acid sequence X1X2X3X4X5GTX8YX 10 DX 12 VX 14 (SEQ ID NO: 209), wherein X1 comprises I or L, X2 comprises W or Y, X3 comprises A, P, or S, X4 comprises D, E, N, Q, R, or S, X5 comprises G, R, or Y, X8 comprises D or Y, and X 10 contains D, E, H, S, or T, and X 12 contains D or S, and X 14contains D, E, or K), (c) the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 LX 12 X 13 X 14 FX 16 X 17 (SEQ ID NO:210), wherein X1 comprises A, D, E, K, N, Q, S, or T; X2 comprises A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X3 comprises I, L, W, or Y; X4 comprises F, T, W, or Y; X5 comprises F, H, or Y; X6 comprises H or Y; X7 comprises D or E; X8 comprises A, S, or V; X9 comprises D, E, H, K, N, Q, or Y; and X 10 contains F, H, or Y, and X 12 contains H or Y, and X 13 contains F or W, and X 14 contains D, I, L, W, or Y, and X 16 contains D or Q, and X 17 contains E, F, H, I, L, M, N, P, W, or Y), (d) the amino acid sequence X1ASX4X5X6X7X8X9LX 11 (SEQ ID NO:211), wherein X1 comprises F or R, X4 comprises E, K, or N, X5 comprises A or G, X6 comprises I, L, or V, X7 comprises A, D, G, L, P, Q, S, V, or Y, X8 comprises K, Q, N, S, or Y, and X9 comprises A, D, E, F, G, H, K, L, N, Q, R, S, or T; and X 11X2 comprises A, G, or S; (e) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence AX2X3X4X5X6X7 (SEQ ID NO: 212), wherein X2 comprises A, D, L, Q, S, T, V, or Y; X3 comprises D, E, K, N, Q, or S; X4 comprises H, I, K, L, M, N, or V; X5 comprises H or L; X6 comprises H, I, L, or M; and X7 comprises D, E, N, S , or T); (f) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence QQX3X4X5X6X7X8T (SEQ ID NO: 213), where X3 comprises G or Y; X4 comprises D, F, G, H, K, L, R, S, T, V, W, or Y; X5 comprises E, K, Q, R, or S; X6 comprises I, F, T, or W; X7 comprises E or P; and X8 comprises L, M, or W.

[0060] In certain embodiments, the anti-NGF binding protein comprises VH-CDR1(GLSLTSX7SMX 10 (SEQ ID NO: 214), wherein X7 comprises A, D, or N; 10 comprises S or V), VH-CDR2 (comprises X1X2SNX5GT (SEQ ID NO: 215), where X1 comprises I or L, X2 comprises W or Y, and X5 comprises G or R), VH-CDR3 (ASIYYYX7AX9YLHWYFDX 12 (SEQ ID NO: 216), wherein X7 comprises D or E, X9 comprises D or E, and X 12 comprises E or F), VL-CDR1 (comprising RASX4GIX7X8NLS (SEQ ID NO: 217), wherein X4 comprises E or K, X7 comprises A, Q, or S, and X8 comprises K or N), VL-CDR2 (comprising AX2X3X4LHS (SEQ ID NO: 218), wherein X2 comprises Q or T, X3 comprises D or S, and X4 comprises I, N, or V), and VL-CDR3 (comprising QQGX4KWPLT (SEQ ID NO: 219), wherein X4 comprises F, W, or Y).

[0061] In certain embodiments, the anti-NGF binding protein comprises one or more (i.e., one, two, three, four, five, or all six) CDRs of fetinized antibody 101 disclosed herein. In certain embodiments, the anti-NGF binding protein comprises one or more (i.e., one, two, three, four, five, or all six) CDRs of affinity matured fetinized antibodies disclosed herein. In certain embodiments, the anti-NGF binding protein comprises CDRs from one or more of fetinized antibody 101 and affinity matured variants provided herein. In certain embodiments, the anti-NGF binding protein comprises the CDRs of the V-type nucleotide sequence shown in FIG. 17A. H In certain embodiments, the anti-NGF binding protein comprises the V CDRs shown in FIG. L In certain embodiments, the anti-NGF binding protein comprises the CDRs of antibody V shown in FIG. H V for Domain H In certain embodiments, the anti-NGF binding protein comprises the CDRs of antibody V shown in FIG. L V for Domain L Contains CDRs.

[0062] According to the invention, in certain embodiments, the anti-NGF binding protein comprises an amino acid sequence of the felinized antibody 101 variant disclosed herein, e.g., V H and / or V. LIn certain embodiments, the anti-NGF binding protein does not include one or more of the amino acid variants listed above. For example, in certain embodiments, the anti-NGF binding protein does not include the arginine of G55R. The amino acid positions are indicated by the residues and numbers of fetinized antibody 101, e.g., S28H is V56H, S30A, S30L, S30P, S30Q, S30V, S30Y, N31Q, S34A, S34G, N53H, N53I, N53K, N53L, N53M, N53V, L54H, H55I, H55L, H55M, S56D, S56E, S56N, S56T. ... H or H at a position corresponding to S28 of antibody 101. The aforementioned positions include CDR and framework amino acid residues.

[0063] In certain embodiments, the anti-NGF binding protein is H and / or V L contains one or more of the following amino acids: S30A, S30Q, N53I, N53V. Pairing of VH and VL chains containing the above sequence mutations demonstrates the compatibility of the VH and VL mutations, as well as the compatibility of the VH and VL domains containing the mutations.

[0064] In certain embodiments, the binding protein comprises a canine antibody or a caninized antibody. In certain embodiments, the binding protein comprises a feline antibody or a felineized antibody.

[0065] In certain embodiments, amino acid residues are mutated to amino acid residues that can preserve the properties of the amino acid side chain. Examples of amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids that contain the following side chains: aliphatic side chains (G, A, V, L, I, P), hydroxyl-containing side chains (S, T, Y), sulfur-containing side chains (C, M), carboxylic acid- and amide-containing side chains (D, N, E, Q), base-containing side chains (R, K, H), and aromatic-containing side chains (H, F, Y, W). The letters in parentheses indicate the one-letter amino acid code. Amino acid substitutions within each group are called conservative substitutions. It is well known that polypeptides containing modified amino acid sequences in which one or more amino acid residues are deleted, added, and / or substituted can retain their original biological activity (Mark D F et al., Proc. Natl. Acad. Sci. USA 81: 5662-5666 (1984); Zoller M J and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. et al., Science 224: 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl. Acad. Sci. USA 79: 6409-6413 (1982)). The number of mutated amino acids is not limited, but is usually within 40%, preferably within 35%, and more preferably within 30% (e.g., within 25%) of the amino acids of each CDR. The identity of the amino acid sequence can be determined as described herein.

[0066] The present invention provides recombinant antibodies that are designed or modified to minimize antigenicity in dogs and cats, hi certain embodiments, the antibodies are further modified to remove T cell epitopes.

[0067] As used herein, the term "dog" includes all domestic dogs, Canis lupus familiaris, or Canis familiaris, unless otherwise indicated.

[0068] As used herein, the term "cat" refers to any member of the Felidae family. Domestic cats, purebred and / or mixed breed companion cats, and wild or feral cats are all Felidae.

[0069] The term "canine framework" or "cat framework" as used herein refers to the amino acid sequences of the heavy and light chains of a canine antibody other than the hypervariable region residues defined herein as CDR residues. For caninized antibodies, in certain embodiments, canine CDRs are identified in canine antibody heavy and light chain variable domain sequences that closely match the CDRs of NGF-binding antibodies from other species. In certain embodiments, the native canine CDRs are replaced in both chains with the corresponding foreign CDRs (e.g., from a rat or mouse antibody). For felineized antibodies, in certain embodiments, feline CDRs are identified in feline antibody heavy and light chain variable domain sequences that closely match the CDRs of NGF-binding antibodies from other species. In certain embodiments, the native feline CDRs are replaced in both chains with the corresponding foreign CDRs (e.g., from a rat or mouse antibody). Optionally, the heavy and / or light chains of the caninized or felineized antibody may contain some mutated or foreign non-CDR residues, such as framework amino acid residues that differ between germline antibody sequences, or mutations that preserve the conformation of the foreign CDR within the antibody.

[0070] There are five major isotypes in dogs (IgA, IgG, IgM, IgD, IgE) and two forms of light chain (kappa and lambda). In dogs, there are four subtypes of IgG: IgGA, IgGB, IgGC, and IgGD (Bergeron et al,2014,Comparative functional characterization of canine IgG subclasses.Veterinary Immunology and Immunopathology.157:31-41). In cats, there are three subtypes of IgG: IgG1a, IgG1b, and IgG2 (Streitzel et al.2014,In vitro functional characterization of feline IgGs.Vet Immunol Immunopathol 158,214-223,doi.org / 10.1016 / j.vetimm.2014.01.012).

[0071] The present invention provides caninized and felinized antibodies modified to modulate one or more effector functions or circulating half-life. The hinge and constant domains of the antibodies bind to host receptors or complement proteins to mediate effector functions and modulate antibody circulation. In certain embodiments, one or more effector functions are enhanced. In certain embodiments, one or more effector functions are reduced or eliminated. In certain embodiments, the antibodies of the present invention comprise modifications to modulate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Non-limiting examples include modification of canine IgGB constant region residues Met242 and / or Leu243 to reduce effector function. In certain embodiments, the IgGB constant region of the present invention comprises M242A and L243A substitutions. In certain embodiments, the second constant domain (CH2) and / or the third constant domain (CH3) comprise mutations and combinations of mutations from wild type designed to modulate binding to the FcRn (neonatal Fc) receptor. In the canine constant region, such mutations include, but are not limited to, substitutions of Ala426, e.g., A426Y or A426H, substitutions of Thr286, e.g., T286L or T286Y, substitutions of Tyr436, e.g., Y436H, and combinations of such mutations, including, but not limited to, A426Y+T286L, A426Y+Y436H, A426H+T286L, and A426H+T286Y. In certain embodiments, a chimeric or caninized antibody of the invention comprises a substitution at amino acid Asn434, e.g., but not limited to, N434H.In the feline constant region, such mutations include, but are not limited to, substitutions at Ser428, including but not limited to S428Y or S428L, substitutions at Gln311, including but not limited to Q311V, substitutions at Leu309, including but not limited to L309V, substitutions at Thr286, including but not limited to T286E, substitutions at Glu380, including but not limited to E380T, and combinations of such mutations, including but not limited to S428Y+Q311V, S428Y+L309V, S428Y+Q311V+T286E, S428Y+Q311V+E380T, and S428Y+L309V+E380T. In a particular embodiment, the chimeric or felinized antibody of the invention comprises substitutions at amino acids Ser428 and / or Ser434, including but not limited to S428L and / or S434H.

[0072] As used herein, the term "antibody" includes antigen-binding fragments of an intact antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any natural, enzymatically accessible, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "specifically binds" or "binds specifically" means that the NGF binding protein of the present invention reacts or associates with NGF more frequently, more rapidly, for a longer duration, and / or with a higher affinity than alternative antigens. For example, the NGF binding protein binds to NGF with substantially higher affinity (e.g., at least 2-fold, or 5-fold, or 10-fold, or 20-fold, or 50-fold, or 100-fold, or 500-fold, or 1000-fold, or 10,000-fold, or more) than other proteins or peptides. In certain embodiments, the NGF binding protein binds to NGF with a higher affinity than its binding epitope or target, e.g., at least 10-fold, or 20-fold, or 50-fold, or 100-fold, or 500-fold, or 1000-fold, or 10,000-fold, or more. -4 M or less, e.g., 10 -5 M, 10 -6 M, 10-7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 Equilibrium dissociation constant K of M D Those skilled in the art will recognize that an antibody that specifically binds to a target (e.g., NGF) from one species may also specifically bind to an ortholog of NGF.

[0073] Antigen-binding fragments of antibodies can be derived, for example, from intact antibody molecules using any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated, chemically or using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate configuration, introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0074] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of an antibody, or the constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the expression "antigen-binding fragment" as used herein.

[0075] In certain embodiments, an antigen-binding fragment of an antibody comprises at least one variable domain. A variable domain may be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in frame with one or more framework sequences. L Domain-associated V H In an antigen-binding fragment having a domain, H and V L The domains may be arranged relative to one another in any suitable configuration. For example, the variable region is a dimer and the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H or V L It may include a domain.

[0076] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(V)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2. (x)V L -C H3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the invention may be linked to each other and / or to one or more monomeric V H or V L The variable and constant domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently associated (e.g., by disulfide bond(s)) with the domains.

[0077] The term "diabody (Db)" refers to a bivalent antibody fragment constructed by gene fusion (e.g., P. Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), EP404,097, WO93 / 11161). In general, a diabody is a dimer of two polypeptide chains. In each polypeptide chain, the light chain variable region (V L ) and the heavy chain variable region (V H ) are connected via a short linker, e.g., a linker of about 5 residues, so that they cannot bind to each other. The linker between the two is too short to allow for V L and V Hcannot form single-chain V region fragments, but instead form dimers. Thus, diabodies have two antigen-binding domains. L Area and V H The domains are linked via a linker of about 5 residues to form V La -V Hb and V Lb -V Ha and then co-expressed, they are secreted as a bispecific Db. The antibody of the present invention may be such a Db.

[0078] Single chain antibodies (also called "scFv") can be prepared by linking the heavy chain V region and the light chain V region of an antibody (for a review of scFv, see Pluckthun "The Pharmacology of Monoclonal Antibodies" Vol. 113, eds. Rosenburg and Moore, Springer Verlag, NY, pp. 269-315 (1994)). Methods for preparing single chain antibodies are known in the art (see, for example, U.S. Patent Nos. 4,946,778, 5,260,203, 5,091,513, and 5,455,030). In such scFvs, the heavy chain V region and the light chain V region are linked to each other via a linker, preferably a polypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 5879-5883). The heavy and light chain V regions in the scFv may be derived from the same antibody or from different antibodies. The peptide linker used to link the V regions may be any single-chain peptide consisting of 12 to 19 residues. The DNA encoding the scFv can be amplified by PCR using a primer pair that defines both ends, using as a template the entire or partial DNA encoding the desired amino acid sequence selected from the DNA encoding the heavy chain or the V region of the heavy chain of the above antibody and the DNA encoding the light chain or the V region of the light chain of the above antibody. Further amplification can then be performed using a combination of the DNA encoding the peptide linker portion and a primer pair that defines both ends of the DNA linked to the heavy chain and the light chain, respectively. After constructing the DNA encoding the scFv, expression vectors containing these DNAs and hosts transformed with these expression vectors can be obtained using conventional methods. Furthermore, the obtained host can be used to obtain scFv according to conventional methods. These antibody fragments can be produced in a host by obtaining genes encoding the antibody fragments and expressing them as outlined above. The modified antibody may be an antibody bound to a variety of molecules, such as polyethylene glycol (PEG).Methods for modifying antibodies have already been established in the art. The "antibody" of the present invention also includes the above-mentioned antibodies.

[0079] The term "Kd" as used herein refers to the dissociation constant of an antibody-antigen interaction. The dissociation constant Kd and the association constant Ka are quantitative measures of affinity. At equilibrium, free antigen (Ag) and free antibody (Ab) are in equilibrium with the antigen-antibody complex (Ag-Ab), and the rate constants ka and kd quantify the rate of each reaction. At equilibrium, ka[Ab][Ag]=kd[Ag-Ab]. The dissociation constant Kd is calculated as Kd=kd / ka=[Ag][Ab] / [Ag-Ab]. Kd has units of concentration, most commonly M, mM, nM, pM, etc. When comparing antibody affinities expressed as Kd, a lower value is indicated for a higher affinity for NGF. The association constant Ka is calculated as Ka=ka / kd=[Ag-Ab] / [Ag][Ab]. Ka has units of reciprocal concentration, most commonly M -1 , mM -1 , nM -1 , pM -1 etc. As used herein, the term "avidity" refers to the strength of antigen-antibody binding, taking into account valency.

[0080] The obtained antibody can be purified to homogeneity. The antibody can be isolated and purified by a method commonly used for protein isolation and purification. For example, the antibody can be isolated and purified by an appropriate combination of one or more methods selected from column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectric focusing (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Such methods are not limited to those listed above. Chromatographic methods include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, and adsorption chromatography. These chromatographic methods can be performed using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. For example, Protein A columns include Hyper D, POROS, and Sepharose FF (Pharmacia). Furthermore, antibodies can be purified by utilizing antigen binding using a carrier on which an antigen is immobilized.

[0081] As used herein, the term "therapeutic agent" refers to any drug or material that has a beneficial effect on a mammalian recipient. Thus, "therapeutic agent" encompasses both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0082] As used herein, "treating" refers to ameliorating at least one symptom of a given disease or condition, curing it, and / or preventing its onset.

[0083] The anti-NGF proteins described herein, including antibodies or fragments thereof, are useful for ameliorating or reducing symptoms of or treating or preventing NGF-related diseases and disorders. The anti-NGF proteins or fragments, as well as combinations with other agents, are administered in the form of pharmaceutical compositions described herein in therapeutically effective amounts to subjects in need of treatment for NGF-related diseases and disorders.

[0084] In certain embodiments, the method includes improving or relieving symptoms of, or treating or preventing pain in a subject.In certain embodiments, anti-NGF protein, antibody, or fragment thereof inhibits the association of NGF with TrkA and / or p75, and is administered, for example, alone or in combination with a second agent, to treat, improve, or relieving symptoms of, or prevent, inflammatory pain, postoperative incision pain, complex cancer pain (including but not limited to primary or metastatic bone cancer pain), fracture pain, osteoporotic fracture pain, osteoporosis pain, burn pain, and other nociceptive pain.

[0085] In certain embodiments, the antibody compositions and methods are used to ameliorate pain, reduce symptoms, treat, or prevent osteoarthritis (OA). OA is a slowly progressive degenerative joint disease characterized by structural changes throughout the joint, including articular cartilage, synovium, subchondral bone, and periarticular components, resulting in pain and loss of joint function. Chronic pain and OA are common in dogs and cats. 20-30% of dogs are clinically affected and have signs of OA. Up to 40% of all cats are clinically affected, and 90% of all cats over the age of 12 have signs of OA.

[0086] The most common site of OA in dogs is the hip, followed by the stifle, shoulder and carpals, whereas in cats the hip, stifle, carpals or spine are most commonly affected.

[0087] The anti-NGF protein, antibody or antibody fragment is optionally administered in combination with one or more active agents, including other analgesics. Such active agents include analgesics, antihistamines, antipyretics, anti-inflammatory agents, antibiotics, antivirals, and anticytokines. Active agents include TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, hepatocyte growth factor (HGF), hepcidin agonists, antagonists, and regulators (including antibodies reactive against any of the foregoing and antibodies reactive against any of their receptors).Active agents also include, but are not limited to, 2-arylpropionic acids, aceclofenac, acemetacin, acetylsalicylic acid (aspirin), alclofenac, aluminoprofen, amoxiprin, ampirone, arylalkanoic acids, azapropazone, benorylate / benolilate, benoxaprofen, bromfenac, carprofen, celecoxib, choline magnesium salicylate, clofezone, COX-2 inhibitors, dexibuprofen, dexiketoprofen, diclofenac, diflunisal, droxicam, ethenzamide, etodolac, etoricoxib, feisulamine, fenamic acid, fenbufen, fenoprofen, flufenamic acid, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indomethacin, indoprofen Also included are kebuzone, ketoprofen, ketorolac, romoxicam, loxoprofen, lumiracoxib, magnesium salicylate, meclofenamic acid, mefenamic acid, meloxicam, metamizole, methyl salicylate, mofebutazone, nabumetone, naproxen, n-arylanthranilic acids, nerve growth factor (NGF), oxametacin, oxaprozin, oxicam, oxyphenbutazone, parecoxib, phenazone, phenylbutazone, phenylbutazone, piroxicam, pirprofen, profen, proglumetacin, pyrazolidine derivatives, rofecoxib, salicylic acid, salicylamide, salicylates, sulfinpyrazone, sulindac, suprofen, tenoxicam, tiaprofenic acid, tolfenamic acid, tolmetin, and valdecoxib.

[0088] Antihistamines can be any compound that opposes the action of histamine or the release of histamine from cells (e.g., mast cells).Antihistamines include, but are not limited to, acrivastine, astemizole, azatadine, azelastine, betastastine, brompheniramine, buclizine, cetirizine, cetirizine analogs, chlorpheniramine, clemastine, CS560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR609, hydroxyzine, levocabastine, loratidine, methscopolamine, mizolastine, norastemizole, phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.

[0089] Antibiotics include amikacin, aminoglycosides, amoxicillin, ampicillin, ansamycin, arsphenamine, azithromycin, azlocillin, aztreonam, bacitracin, carbacephems, carbapenems, carbenicillin, cefaclor, cefadroxil, cephalexin, cephalothin, cephamandole, cefazolin, cefdinir, cefditoren, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, and ceftizoxime. ceftobiprole, ceftriaxone, cefuroxime, cephalosporin, chloramphenicol, cilastatin, ciprofloxacin, clarithromycin, cylindromycin, cloxacillin, colistin, cotrimoxazole, dalfopristin, demeclocycline, dicloxacillin, dirithromycin, doripenem, doxycycline, enoxacin, ertapenem, erythromycin, ethambutol, flucloxacillin, fosfomycin, furazolidone, fusidic acid, gatifloxacin, geldanamycin, gentamicin, glycopenem, putide, herbimycin, imipenem, isoniazid, kanamycin, levofloxacin, lincomycin, linezolid, lomefloxacin, loracarbef, macrolides, mafenide, meropenem, methicillin, metronidazole, mezlocillin, minocycline, monobactam, moxifloxacin, mupirocin, nafcillin, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oxacillin, oxytetracycline, paromomycin, penicillin, penicillins, piperacillin, platensimycin, po Rimyxin B, polypeptides, prontosil, pyrazinamide, quinolones, quinupristin, rifampicin, rifampin, roxithromycin, spectinomycin, streptomycin, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, sulfonamide, teicoplanin, telithromycin, tetracycline, ticarcillin, tinidazole, tobramycin, trimethoprim, trimethoprim-sulfamethoxazole, troleandomycin, trovafloxacin,and vancomycin.

[0090] Active agents also include aldosterone, beclomethasone, betamethasone, corticosteroids, cortisol, cortisone acetate, deoxycorticosterone acetate, dexamethasone, fludrocortisone acetate, glucocorticoids, hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids, and triamcinolone. Any suitable combinations of these active agents are also contemplated.

[0091] The current most common treatment for OA and OA-related pain is NSAIDs (also anti-analgesic drugs). NSAIDs are not always fully effective, usually need to be administered daily, and none are approved for long-term use in cats in the United States. Furthermore, there are concerns regarding safety and tolerability when using NSAIDs in both dogs and cats, especially for long-term treatment. Coadministration of NSAIDs with anti-NGF mAbs for extended periods is not recommended.

[0092] In certain embodiments, treatment includes co-administration of dietary supplements containing omega-3 fatty acids, microlactin, and / or glucosamine / chondroitin to aid in joint health. Adequan (a polysulfated glycosaminoglycan), an FDA-approved disease-modifying drug that inhibits cartilage loss, may also be co-administered.

[0093] Formulations and Methods of Administration

[0094] When used in vivo, the therapeutic agent described herein is generally incorporated into a pharmaceutical composition prior to administration. In such compositions, one or more therapeutic compounds described herein are present as active ingredient(s) (i.e., present at a level sufficient to produce a statistically significant effect on the symptoms of cystic fibrosis as measured using a representative assay). A pharmaceutical composition comprises one or more such compounds in combination with any pharma- ceutically acceptable carrier(s) known to those skilled in the art to be suitable for a particular mode of administration. In addition, other pharma- ceutical active ingredients (including other therapeutic agents) may be present in the composition, but are not required to be present.

[0095] The antibodies of the present invention can be formulated according to standard methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA) and may include pharma- ceutically acceptable carriers and / or excipients. The present invention relates to compositions (including reagents and pharmaceuticals) comprising the antibodies of the present invention and pharma- ceutically acceptable carriers and / or excipients. Exemplary carriers include surfactants (e.g., PEG and Tween), excipients, antioxidants (e.g., ascorbic acid), colorants, flavorings, preservatives, stabilizers, buffers (e.g., phosphate, citric acid, and other organic acids), chelating agents (e.g., EDTA), suspending agents, isotonicity agents, binders, disintegrants, lubricants, glidants, and flavoring agents. However, the carriers that may be used in the present invention are not limited to this list. In practice, other commonly used carriers can be appropriately used, such as light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The composition may also contain other low molecular weight polypeptides, proteins, such as serum albumin, gelatin, and immunoglobulins, as well as amino acids, such as glycine, glutamine, asparagine, arginine, and lysine. When the composition is prepared as an aqueous solution for injection, it may comprise an isotonic solution including, for example, saline, glucose, and other adjuvants including, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, which may also contain suitable solubilizing agents, for example, alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol and PEG), and non-ionic surfactants (polysorbate 80 and HCO-50).

[0096] If necessary, the antibody of the present invention may be encapsulated in a microcapsule (a microcapsule made of hydroxycellulose, gelatin, polymethylmethacrylate, etc.) and used as a component of a colloidal drug delivery system (liposome, albumin microsphere, microemulsion, nanoparticle, and nanocapsule) (see, for example, "Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980)). Furthermore, methods for producing sustained-release drugs are also known and can be applied to the antibody of the present invention (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:98-105 (1982); U.S. Patent No. 3,773,919; European Patent Application No. 58,481; Sidman et al., Biopolymers 22:547-556 (1983); EP:133,988).

[0097] The preferred route of administration in both dogs and cats is by subcutaneous injection, usually into the skin at the base of the neck. In certain embodiments, the anti-NGF protein is packaged in an integrated delivery system, such as a pen or prefilled syringe for subcutaneous administration. Ghil et al. describe the administration of adalimumab biosimilar SB5 via a prefilled syringe (PFS) and an autoinjector (AI) pen based on injection site pain, patient preference, and safety in rheumatoid arthritis (RA) (see Ghil et al., Usability and safety of SB5 (an adalimumab biosimilar) prefilled syringe and autoinjector in patients with rheumatoid arthritis. Curr Med Res Opin 2019 Mar;35(3):497-502). The compositions of the present invention are administered to dogs, cats, and other mammals alike.

[0098] With respect to the treatment of a disease state / condition, the term "therapeutically effective amount" refers to an amount of a compound, either alone or contained in a pharmaceutical composition, that when administered as a single dose or multiple doses, can have a detectable positive effect on any symptom, aspect, or characteristic of the disease state / condition. Such an effect need not be absolutely effective.

[0099] As used herein, the terms "treat," "treating," and "treatment" include administration of a compound prior to the onset of clinical symptoms of a disease state / condition to prevent symptoms, as well as administration of a compound after the onset of clinical symptoms of a disease state / condition to reduce or eliminate any symptoms, aspects, or characteristics of the disease state / condition. Such treatment need not be absolutely beneficial.

[0100] In certain embodiments, the therapeutic agents may be administered systemically, for example orally, in combination with a pharma- ceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For oral therapeutic administration, the active compounds may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be from about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0101] Tablets, troches, pills, capsules, etc. may contain: binders such as gum tragacanth, acacia, corn starch, or gelatin, excipients such as dicalcium phosphate, disintegrating agents such as corn starch, potato starch, alginic acid, lubricants such as magnesium stearate, and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, in addition to the above types of materials, it may contain a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or to modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, etc. Syrup or elixir may contain active compound, sucrose or fructose as sweetener, methylparaben and propylparaben as preservatives, dyes and flavorings such as cherry or orange flavor.Of course, any material used to prepare unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts used.Furthermore, active compound may be incorporated into sustained-release preparations and devices.

[0102] The active compound may also be administered intravenously or intraperitoneally by infusion or injection.The solution of the active compound or its salt may be prepared in water, optionally mixed with a non-toxic surfactant.Dispersions may also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, and in oils.These preparations contain a preservative to prevent the growth of microorganisms under normal storage and use conditions.

[0103] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, optionally encapsulated in liposomes, suitable for the extemporaneous preparation of sterile injection or infusion solutions or dispersions. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be, for example, a solvent or liquid dispersion medium, including water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. The inhibition of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0104] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in a suitable solvent with various other ingredients mentioned above, and then optionally sterilizing by filtration. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which yields a powder of the active ingredient and any additional desired ingredient present in a previously sterile-filtered solution.

[0105] The useful dose of the compounds of the present invention can be determined by comparing their in vitro activity with their in vivo activity in animal models. In certain embodiments, the useful dose is about 0.1 mg / kg to about 5 mg / kg, or about 0.5 mg / kg to about 2 mg / kg. Methods for extrapolating effective doses in humans and animals of different sizes are known in the art, see, for example, U.S. Patent No. 4,938,949.

[0106] The amount of the compound, or an active salt or derivative thereof, required for therapeutic use will vary depending not only on the particular salt chosen, but also on the route of administration, the nature of the condition being treated, and the age and condition of the patient, and is ultimately at the discretion of the attending physician or clinician.

[0107] In general, however, a suitable dosage is in the range of about 0.5 to about 100 mg / kg body weight per day, e.g., about 10 to about 75 mg / kg body weight, e.g., 3 to about 50 mg / kg of recipient body weight / day, preferably in the range of 6 to 90 mg / kg / day, and most preferably in the range of 15 to 60 mg / kg / day.

[0108] The compound is conveniently administered in unit dosage form, for example containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently 50 to 500 mg of active ingredient per unit dosage form.

[0109] Ideally, the active ingredient should be administered to achieve a peak plasma concentration of the active compound of about 0.5 to about 75 μM, preferably about 1 to 50 μM, and most preferably about 2 to about 30 μM. This may be achieved, for example, by intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 to 100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion providing about 0.01 to 5.0 mg / kg / hour, or by intermittent infusions containing about 0.4 to 15 mg / kg of the active ingredient(s).

[0110] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, two, three, four or more sub-doses per day. The sub-dose itself may be further divided, for example, into a number of discrete loosely spaced administrations.

[0111] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

[0112] The present invention is further illustrated in the following examples, which are provided for illustrative purposes and are not intended to limit the invention in any manner. EXAMPLES

[0113] Example 1 Generation and characterization of rat antibody 2166, which binds canine NGF.

[0114] Lewis rats were immunized with human NGF (R&D Systems, 256-GF-100 / CF) every week for 8 weeks. Titers were measured in a flow cytometry assay using human NGF-coated beads. Beads were complexed with human NGF (R&D Systems, 256-GF-100 / CF) and incubated with different dilutions of serum (1:100, 1:500, 1:2500) for 30 min. Beads were washed and binding was detected using a fluorescently labeled anti-rat IgG secondary antibody. Fluorescence was measured using Intellicyt iQue Screener Plus. Titers were measured at 1:2500 dilution for all three rats, which were approximately 100-fold higher than the values ​​in normal Lewis rat serum.

[0115] Lymph nodes from femoral, tibial, and pelvic bones (brachial, axillary, inguinal, popliteal, and ischial) as well as bone marrow were harvested from rats with significant NGF titers. Cells were isolated from both tissues and enriched for plasma cells using flow cytometry. The enriched plasma cell suspension was injected into AbCellera's microfluidic screening device, which contains separate reaction chambers of 91,000 or 153,000 nanoliter volume. Single cells secreting NGF-specific antibodies were identified and isolated using a bead-based assay. Beads coated with anti-rat IgG antibodies were flowed over the microfluidic screening device and incubated with single antibody-secreting cells. IgG secreted by plasma cells was captured on the beads using the constant region. Fluorescently labeled human NGF antigen was then used to assess binding to the secreted IgG immobilized on the beads. Positive hits were identified using machine vision and retrieved using an automated robot-based protocol. Approximately 269,000 individual B cells were screened in the NGF-binding assay, and 592 cells expressed antibodies that recognized NGF. From these positive cells, 190 unique antibody sequences were identified. From the 190 antibodies, 88 antibodies were selected based on clonotype diversity.

[0116] NGS sequencing libraries (MiSeq, Illumina) were generated using an automated workstation (Bravo, Agilent) by single-cell polymerase chain reaction and custom molecular biology protocols. Sequence data was analyzed using a custom bioinformatics pipeline to obtain paired heavy and light chain sequences for each antibody-secreting cell recovered (Jones et al., 2020, bioRxiv 2020.09.30.318972.doi:10.1101 / 2020.09.30.318972). The amino acid sequences of the heavy and light chain variable domains of rat antibody 2166 are shown in Figure 1 and Figure 2, respectively, with the CDRs indicated. The variable (V(D)J) regions of each antibody chain were synthesized and inserted into mammalian expression plasmids using a custom automated high-throughput cloning pipeline.

[0117] Expression vectors were transfected into Expi293-F cells (Gibco, ThermoFisher Scientific) in 24 deep-well plates using the manufacturer's recommended protocol. Four days after transfection, conditioned medium was purified with Protein A beads, and antibodies were eluted by adding 100 mM glycine, pH 2.0, and neutralized to pH 7.0 by adding 1 M Tris-HCL, pH 8.0. Neutralized antibodies were buffer exchanged into PBS, pH 7.2.

[0118] Analysis of the purified antibodies included CE-SDS (denaturing capillary sodium dodecyl sulfate gel electrophoresis) and DSF (differential scanning fluorescence). Purity of the purified antibodies was determined using CE-SDS and was completed using a LabChip GXII Touch instrument (Perkin Elmer). Two microliters of antibody solution at a concentration of 350 μg / mL in PBS was mixed with non-reducing denaturing buffer (Perkin Elmer) and incubated at 70°C for 10 minutes. Separation and detection were performed using the HT Antibody Analysis 200 assay setting on the LabChip instrument (Perkin Elmer). Fluorescence data was analyzed by percentage purity using LabChip GX Reviewer Software (Perkin Elmer). The percentage purity of rat monoclonal antibody 2166 was 96%.

[0119] The melting temperature (Tm) of the antibodies was assessed by differential scanning fluorimetry (DSF) using a SYPRO™ Orange fluorescent probe (5000x concentrated solution, Thermo Fisher Scientific). 6 μL of 350 μg / mL mAb solution in PBS was mixed with 6 μL of 19x concentrated SYPRO™ Orange solution diluted in PBS. Thermal denaturation, assessed by change in fluorescence, was measured in a Bio-Rad C1000 Touch Thermal Cycler instrument (Bio-Rad Laboratories) using a CFX96 Real-Time System reader head (Bio-Rad Laboratories). The excitation and emission wavelengths were 450-490 nm and 560-580 nm, respectively. The fluorescent signal was measured at a starting temperature of 25°C, incremented by 0.5°C / min, up to 95°C. Data was analyzed and melting curves were integrated using Bio-Rad CFX Maestro software (v1.1). The Tm was defined as the minimum obtained from the derivative of the melting curve. The Tm of rat antibody 2166 was 66.5°C.

[0120] Binding assays were completed to confirm antibody binding to NGF (R&D Systems, 256-GF-100 / CF). Additionally, antibody specificity was determined by testing antibody binding to closely related proteins, NT-3 and BDNF. A multiplex bead assay on a high-throughput flow cytometer was used to confirm that unique antibody sequences bound to the screening targets. Different optically encoded beads were complexed with either human NGF (R&D Systems, 256-GF-100 / CF), NT-3 (R&D Systems, 267-N3-025 / CF), or BDNF (R&D Systems, 248-BDB-050 / CF). Purified antibodies were incubated with multiplexed beads at different antibody concentrations for 30 minutes at room temperature. Beads were washed and binding was detected using fluorescently labeled secondary antibodies. Fluorescence was measured using high-throughput plate-based flow cytometry on an Intellicyt® iQue Screener Plus.

[0121] The median fluorescence intensity of each antibody was normalized to the median fluorescence intensity of the appropriate isotype control for each individual bead type. Antibody values ​​greater than 10-fold higher than the isotype were considered binders.

[0122] Antibody 2166 bound NGF at greater than 59-fold above background levels, and binding of this antibody to NT-3 and BDNF was at background levels.

[0123] A functional assay with TF-1 cells was used to determine whether binding of the 2166 antibody to canine NGF blocks the ability of canine NGF to induce signaling through human TrkA, the high affinity receptor for NGF (Chevalier et al., 1994. Blood, 83:1479). In these studies, canine NGF (Genbank NP_001181879.1) was used as the source of NGF. Canine NGF with a C-terminal strep tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) was stably expressed in Dmel-2 cells and purified using StrepTactinXT chromatography followed by a polishing step with Superdex 200 16 / 600 chromatography. The proliferation of TF-1 cells can be stimulated by various growth factors, such as GM-CSF and NGF. TF-1 cells (ATCC-CRL2003) were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / ml streptomycin, and 2 ng / mL recombinant human GM-CSF. The cells were cultured at a concentration of 3 × 10 4 ~5×10 5 Viable cells / mL were maintained and passaged every 48 hours. Each condition was performed in triplicate wells. Cells were harvested and counted. Cells were cultured at 1.75 x 10 5 Cells were then resuspended at 0.5×10 cells / ml and incubated in flasks in a humidified 37° C., 5% CO2 incubator for 4 hours. During incubation, NGF / antibody mixtures were prepared in media as 2× media solutions in complete media without GM-CSF and complete media with 10 ng / mL canine NGF. Antibodies were added to the appropriate 2× media solution and incubated at room temperature for at least 1 hour before adding the NGF / antibody solution to the cells. Cells were then harvested and cultured at 0.5×10 cells / ml in media without GM-CSF. 6Cells were resuspended in the appropriate medium volume to achieve a suspension of cells / ml. 50 μl of cell suspension was added per well of a 96-well plate, to which 50 μl of 2×NGF / antibody medium was added per well of the cell plate. Cells were incubated for 48 hours in a humidified 37° C., 5% CO2 incubator, then 20 μl of Aqueous One solution Reagent (Promega) was added per well. Cells were incubated for an additional 4 hours in a humidified 37° C., 5% CO2 incubator, then absorbance was read at 490 nm in a BioTek Synergy / neO2. Data was analyzed by subtracting blank wells from all measurements. Percent inhibition was calculated using the following formula: Percent inhibition (%)=100×[1-(X-MIN) / (MAX-MIN)], where X=signal at a given concentration, MAX=0% inhibition=canine NGF only, and MIN=100% inhibition=no NGF control. For each condition, the average was calculated in triplicate. Proliferation data for rat antibody 2166 and the isotype rat antibody control are shown in Figure 3. The data show that rat antibody 2166 effectively blocks NGF binding to TrkA.

[0124] The VH domain of antibody 2166 was fused to a canine IgGB constant domain (Tang et al. 2001. Vet. Immunol. Immunopathol. 80:259) and the VL domain of antibody 2166 was fused to a canine kappa constant domain to generate a canine chimeric antibody (Figure 4). Two residue changes (AA) (underlined and bold) were made in the Fc to eliminate effector activity, and these changes are similar to the "LALA" mutations described for human IgG1 Fc (Tamm & Schmidt, 1997. Int. Rev. Immunol. 16:57). These two constructs were subcloned into pcDNA3.4 (ThermoFisher Scientific), co-transfected using the Expi293 system (ThermoFisher Scientific), and purified by HiTrap Protein A HP chromatography. The purity of the antibody was determined to be >95% by SDS / PAGE, and the monomeric fraction of the antibody was determined to be 98% by SEC (size exclusion chromatography).

[0125] The affinity of the canine 2166 chimeric antibody to canine NGF was measured by SPR (surface plasmon resonance). For these studies, canine NGF (Genbank NP_001181879.1) was produced by fusing a Flag tag (DYKDDDDK) at the C-terminus, expressing the canine NGF construct with baculovirus technology, and then purifying the NGF with anti-DYKDDDDK G1 affinity chromatography. The binding kinetics of the canine 2166 chimeric antibody to canine NGF was measured using a Biacore T200 instrument. The assay format was to capture the Fc of the 2166 antibody on a protein A sensor chip and use canine NGF as the analyte. The running buffer was HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% tween20) and the instrument temperature was set at 25°C. The flow rate was 40 μl / min and the concentrations of the five analytes tested in duplicate ranged from 0.78 nM to 12.5 nM. The binding signals were corrected for blanks and the resulting sensorgrams (Figure 5) were used to calculate the rate constants (k a and k d ) and binding affinity (K D Under these binding conditions, the binding rate of the canine 2166 chimeric antibody was determined to be k a (1 / Ms)=1.2E+7, k d (1 / s)=3.5E-6, and K D = 3E-13. The measured affinity (K D ) is beyond the sensitivity of the Biacore instrument but is estimated from these data to be at least 50 pM.

[0126] The ability of the canine 2166 chimeric antibody to block canine NGF binding to the canine NGF receptors (TrkA and p75) was measured in an SPR assay on a Biacore T200. The assay format was to capture the NGF receptor on a sensor chip and flow either canine NGF alone, NGF mixed with canine 2166 chimeric antibody, or canine 2166 chimeric antibody alone.

[0127] The NGF receptors used in the assay consist of the extracellular domains of canine p75 (XP_038340439.1) and canine TrkA (XP_038398906.1) fused to human IgG1 Fc (UniProtKB P01857) with a 2xGly-Gly-Gly-Ser linker between the receptor and the Fc. The fusion proteins were expressed in CHO cells and purified by Protein A chromatography. In the assay, the proteins p75-Fc and Trk-Fc were captured on a human anti-Fc sensor chip.

[0128] The analytes were NGF alone, canine 2166 chimeric antibody alone, and canine 2166 chimeric antibody-NGF mixture (molar ratio 2:1). The seven concentrations of NGF in the NGF-only condition ranged from 0.78 nM to 50 nM. The concentrations of canine NGF in the canine 2166 chimeric antibody-NGF mixture were 50 nM, 25 nM, and 12.5 nM. Finally, the four concentrations of the canine 2166 chimeric antibody alone condition ranged from 12.5 nM to 100 nM. The temperature and flow rate of the device were set at 25°C and 40 μL / min, respectively.

[0129] The binding signals were corrected for a reference and the resulting sensorgrams were used to calculate the rate constants (k a and k d ) and binding affinity (K D ) was determined. Sensorgrams (FIGS. 5, 6, 7, 8) represent the NGF-only condition and the canine 2166 chimeric antibody-NGF mixture for both the p75-Fc and TrkA-Fc receptors. Under these binding conditions, the binding rate of canine NGF alone to the dimeric form of canine p75 was k a (1 / Ms)=1.2E+10, k d (1 / s)=45, and K D = 3.7E-9 (Figure 6). The binding rate of canine NGF alone to the dimeric form of canine TrkA was k a (1 / Ms)=1.3E+7, k d (1 / s)=1.7E-4, and K D= 1.3E-11 (Figure 7). In the condition of canine 2166 chimeric antibody alone, no binding to the canine NGF receptor was observed (data not shown). As shown by the sensorgrams (Figures 8 and 9), canine 2166 chimeric antibody effectively blocks the binding of canine NGF to canine TrkA and p75.

[0130] Example 2 Caninization of rat 2166 antibody

[0131] A canine antibody database was generated by performing NGS (next generation sequencing) on ​​canine PBMCs (peripheral blood mononuclear cells). The database contained 5.0 × 10 6 VH domains, 3.7 × 10 6 VK domains, and 2.6 × 10 6 The sequences of the VL domains are included. The HCDR1,2 and LCDR1,2,3 sequences of the 2166 parent antibody were used in an algorithm to identify the closest canine CDR sequences and their linked framework sequences in the canine antibody database. These linked framework sequences were included in an scFv phage display library along with the closest framework germline sequence and the linked framework sequence with 1-3 residues reverted to the closest germline. A proprietary algorithm was used to identify a set of CDR sequences that are similar to the original 2166 CDRs and have closer identity to the germline and expressed CDR sequences. These CDR and framework sequences were used to identify a set of CDR sequences with a theoretical complexity of 3×10 12A scFv antibody phage display library of was generated. Selection of antibody phages was completed in four rounds using canine NGF, with stringency increasing each round by decreasing the antigen concentration and increasing the number of washes. Specifically, 96 multi-well plates were coated with 200 pmol NGF in the first round, 100 pmol in the second round, and 50 pmol in the third and fourth rounds. The number of washes with PBS-tween20 (0.01%) after selection was 6 times in the first round, 7 times in the second round, 8 times in the third round, and 9 times in the fourth round. The scFv clones output from the third and fourth rounds were sequenced, and unique clones were reformatted into IgG and screened for binding to canine NGF by SPR. The sequences and binding kinetics to canine NGF of the top 69 caninized clones along with the parent clone 2166 are shown in Figures 1 and 2 and Table 2. [Table 2-1] [Table 2-2]

[0132] Sensorgrams of all 69 clones are shown in Figure 10. SPR was completed by amine coupling of the antibody (approximately 5 μg / ml) to a HC30M sensor chip by EDC / NHS activation followed by quenching with ethanolamine HCL. Canine NGF (Genbank NP_001181879.1) used as analyte for SPR analysis was the same preparation as described in Example 1 for C-terminal Flag-tagged (DYKDDDDK) canine NGF.

[0133] The ability of caninized SC42_101 antibody to block canine NGF binding to canine NGF receptors (TrkA and p75) was measured in an SPR assay on a Biacore T200. The assay format was to capture the NGF receptor on a sensor chip and flow either canine NGF alone, NGF mixed with caninized SC42_101 antibody, or caninized SC42_101 antibody alone. The receptor blocking method was identical to that described for the canine 2166 chimeric antibody in Example 1. The sensorgrams (Figures 11, 12, 13, 14) represent the NGF only condition and the caninized SC42_101 antibody-NGF mixture for both the p75-Fc and TrkA-Fc receptors. Under these binding conditions, the binding rate of canine NGF alone to the dimeric form of canine p75 was k a (1 / Ms)=3.8E+7, k d (1 / s)=0.1, and K D = 2.7E-9 (Figure 11). The binding rate of canine NGF alone to canine TrkA in the dimeric form was k a (1 / Ms)=2.4E+7, k d (1 / s)=1.6E-4, and K D = 6.6E-12 (Figure 12). Under the condition of caninized SC42_101 antibody alone, no binding to the canine NGF receptor was observed (data not shown). As shown by the sensorgrams (Figures 13 and 14), the caninized SC42_101 antibody effectively blocks the binding of canine NGF to canine TrkA and p75.

[0134] Example 3 Feline version of rat 2166 antibody

[0135] A feline antibody database was generated by performing NGS (next generation sequencing) on ​​feline PBMCs (peripheral blood mononuclear cells). The database contains 7.5 × 10 6 VH domains, 1.3 × 10 6 VK domains, and 3.8 × 10 6The sequences of VL domains are included. The HCDR1,2 and LCDR1,2,3 sequences of the 2166 parent antibody were used in an algorithm to identify the closest feline CDR sequences and their linked framework sequences in the feline antibody database. These linked framework sequences were included in an scFv phage display library along with the closest framework germline sequence and linked framework sequences with 1-3 residues reverted to the closest germline. A proprietary algorithm was used to identify a set of CDR sequences that are similar to the original 2166 CDRs and have closer identity to the germline and expressed CDR sequences. These CDR and framework sequences were used to identify a set of CDR sequences with a theoretical complexity of 3×10 12 An scFv antibody phage display library was generated. Since the processed form of feline NGF (XP_004001166.1) is identical to the processed form of canine NGF (NP_001181879.1), canine NGF tagged at the C-terminus with the Flag tag (DYKDDDDK) described in Example 1 was used in the felineization test.

[0136] Selection of antibody phage was completed in four rounds with NGF, with stringency increasing each round by decreasing the antigen concentration and increasing the number of washes. Specifically, 96 multi-well plates were coated with 200 pmol of NGF in the first round, 100 pmol in the second round, and 50 pmol in the third and fourth rounds. The number of washes with PBS-tween20 (0.01%) after selection was 6 times in the first round, 7 times in the second round, 8 times in the third round, and 9 times in the fourth round. The scFv clones output from the third and fourth rounds were sequenced, and unique clones were reformatted into IgG and screened for binding to NGF by SPR. The variable domain of clone 101 is shown in Figure 15, with the CDR regions underlined. The affinity of felineized clone 101 to NGF was measured by SPR. The assay format was to immobilize goat anti-cat IgG (30 μg / ml) on a Series S CM5 biosensor using EDC / NHS and quench remaining sites with ethanolamine. Felineated Clone 101 (1 μg / ml) was captured on the goat anti-cat IgG sensor chip. Five concentrations of NGF were captured using single cycle kinetics. The binding kinetics of felineated Clone 101 to NGF was k a (1 / Ms)=3.8E+5, k d (1 / s)=3E-3, and K D =7.8E-9.

[0137] Example 4 Affinity maturation of feline clone 101 using site-directed mutagenesis of the CDRs

[0138] In the first affinity maturation approach, the heavy chain variable and CH1 domains of feline clone 101 (Table 3) were subcloned into a GenScript FASEBA plasmid. This construct contained a single domain antibody against serum albumin (SASA) tag with low pM affinity for albumin at the C-terminus of the heavy chain (VH-CH1) (see, e.g., US2013 / 0129727A1) and further downstream a His tag for purification. The light chain variable domain was subcloned into a proprietary E. coli expression vector using feline Cκ (Table 3). Both the heavy and light chains had a PelB (pectate lyase B) signal peptide at the N-terminus to facilitate secretion of the Fab when expressed in TG1 E. coli. Expression of the variable domains was regulated by the Lac promoter. [Table 3]

[0139] GenScript's proprietary Precision Mutant Library (PML), which utilizes semiconductor-based oligonucleotide synthesis technology, was used to generate variant libraries for each CDR position within the heavy and light chains. For mutant generation, a combination of Kabat and IMGT methodologies was used to define the CDRs, and the residues selected for each CDR are shown in Table 4 below. The CDR residue numbers are shown in brackets. [Table 4]

[0140] The quality of the libraries was verified using NGS (Next Generation Sequencing). 44 PML clones from each library were selected for expression in E. coli in 96 deep-well plates by plating on 2YT medium and inducing with 0.2 mM IPTG overnight at room temperature. Fabs secreted into the medium were analyzed for binding activity by completing an ELISA. In this ELISA, plates were coated with 10 μg / ml BSA overnight at 4° C., washed three times with PBS containing 0.1% Tween 20, pH 7.4 (PBST), blocked non-specific interactions for 1 h at 37° C. in PBS (phosphate buffered saline, pH 7.4) containing 3% non-fat dry milk, washed three times with PBST, added crude Fab supernatant (diluted 1:1 in PBST), incubated for 1 h at 37° C., washed three times with PBST, added 0.15 μg / ml NGF, incubated for 1 h at 37° C., washed three times with PBST, added horseradish peroxidase (HRP)-conjugated anti-Flag tag antibody (Flag tag present on NGF), incubated for 45 min at room temperature, washed three times with PBST, incubated with TMB substrate for 10 min at room temperature, and detected the HRP complex by measuring absorbance at 450 nm. The top 100 clones with clear increases in affinity as measured by ELISA were sequenced to detect variants within the CDRs, identifying 57 unique clones. The mutations in each of the 57 clones from clone 101 are summarized in Table 5. [Table 5]

[0141] The binding of 57 unique clones was confirmed by dissociation rate screening assay in SPR assay performed on Biacore T200. For SPR analysis, bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. The sensor chip surface was activated with 50 mmol / L H-hydroxysuccinimide and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for 420 seconds. Then, BSA diluted in 10 mM sodium acetate, pH 4.5, was injected. After the amine coupling reaction, the remaining active coupling sites on the chip surface were blocked with 1 mM ethanolamine hydrochloride. The selected Fab-SASA variants in the conditioned medium were captured on the BSA-coated chip. The running buffer was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween20, pH 7.4). After equilibration, antigen was injected for 120 seconds (association phase), followed by injection of running buffer for 420 seconds (dissociation phase). Dissociation rates of Fab-SASA clones were obtained by locally fitting the experimental data to a 1:1 interaction model using Biacore T200 evaluation software. Fab variants were ranked by dissociation rate constants (dissociation rate, kd) shown in Table 6. [Table 6-1] [Table 6-2]

[0142] Fab variants G55R, S30A, S30Q, S35V, N53I, F112E, S97Q, and N53V were selected for combinatorial library construction (Table 7). [Table 7]

[0143] The combinatorial library was constructed in the same Fab-SASA vector as above. The theoretical diversity of the combinatorial library is 2 × 2 × 2 × 3 × 3 = 144, and the size of the constructed library is 5.6 × 10 7 CFU (colony forming units). The in-frame rate and diversity of the library were assessed by DNA sequencing, and the results are shown in the table below. [Table 8] [Table 9] [Table 10]

[0144] 184 clones were randomly selected from the combinatorial library and completed binding by NGF ELISA. NGF ELISA was the same method as above. The top 20 clones in ELISA binding were sequenced and tested in SPR dissociation rate assay. The binding results are shown in Table 11 for each amino acid combination. Table 12 shows the variable domain sequence IDs of the top 20 clones. The pairing of VH and VL shows that the mutations of VH and VL are substantially compatible, and the VH and VL domains are compatible. [Table 11] [Table 12]

[0145] Example 5 Affinity maturation of feline clone 101 by scFv phage display

[0146] In the second affinity maturation approach, an scFv phage display library was constructed containing the following sequences for the felinized clone 101 framework and the heavy and light chain CDR sequences shown in Table 13: [Table 13-1] [Table 13-2] [Table 13-3]

[0147] The library diversity of the heavy chain was 37 (HCDR1) × 11 (HCDR2) × 57 (HCDR3) = 23,199, and the library diversity of the light chain was 24 (LCDR1) × 13 (LCDR2) × 22 (LCDR3) = 6,864. The library diversity of the combined heavy and light chain was 1.59 × 10 8Selection of antibody phages was completed in five rounds using NGF, with stringency increased by decreasing the antigen concentration and increasing the number of washes in each round. Specifically, 96 multi-well plates were coated with 200 pmol of NGF in the first round, 50 pmol of NGF in the second and third rounds, 25 pmol in the fourth round, and 10 pmol in the fifth round. The number of washes with PBS, pH 7.4-Tween 20 (0.01%) after the selection steps was 3 times after the first round, 4 times after the second round, 5 times after the third round, 6 times after the fourth round, and 7 times after the fifth round. 760 clones of phages were isolated from each output of the third, fourth, and fifth rounds screened by NGF-binding ELISA. Positive clones were sequenced, and 140 unique positive clones were reformatted into feline IgG1a, expressed in CHO cells, and purified with protein A. SPR was completed by amine coupling of the antibody (approximately 5 μg / ml) to a HC30M sensor chip by EDC / NHS activation followed by quenching with ethanolamine HCL. NGF was analyte diluted in HEPES buffered saline containing 0.01% tween20 and 0.5 mg / ml BSA. NGF was run at concentrations of 500 nM, 166 nM, 55 nM, 18 nM, 6.2 nM, 2.0 nM, 0.68 nM, and 0.23 nM. The affinities of the top three affinity matured clones are shown in Table 14 below. The sequences of the variable domains of the top three clones (SC-184_76, SC-184_102, SC-184_110) are shown in Figure 17. [Table 14]

[0148] Example 6 SPR and NGF receptor blocking data of affinity matured feline antibodies against feline NGF

[0149] The affinity matured antibodies AHF17602, SC-184_76, SC-184_102, and SC-184_110 described in the first affinity maturation approach, as well as the latter three clones containing the G55R mutation (SC-184_76-Arg, SC-184_102-Arg, and SC-184_110-Arg), were evaluated for affinity to NGF using SPR with a Biacore T200 instrument. The variable domain sequences of AHF17602, SC-184_76-Arg, SC-184_102-Arg, and SC-184_110-Arg are shown in Figure 17. In addition, clone 101 was also evaluated. Antibodies were captured using an anti-cat coupled CM5 chip. NGF binding was then assessed at multiple concentrations starting at 50 nM using PBSP+running buffer (Cytiva) at a flow rate of 30 μL / min. Binding time lengths were 120 seconds and dissociation times were run at 600 seconds. The chip surface was regenerated with 10 mM glycine. Reference-subtracted sensorgrams were fitted to a 1:1 binding model using Biacore T100 Evaluation software. The data are shown in Table 15 below and the sensorgrams are shown in Figure 18. [Table 15]

[0150] For NGF receptor blocking experiments, feline TrkA and p75 NGF receptors were produced and used in SPR experiments using a Biacore T200. The extracellular domain of feline TrkA (XP_023103311) was cloned with an AviTag (GLNDIFEAQKIEWHE) and 8xHis tag at the C-terminus and expressed in HEK293 cells. Recombinant feline TrkA protein was purified from conditioned medium using nickel chromatography. The extracellular domain of feline p75 (XP_023099534) was cloned with an AviTag (GLNDIFEAQKIEWHE) and 8xHis tag at the C-terminus and expressed in HEK293 cells. Recombinant feline p75 protein was purified from conditioned medium using nickel chromatography. Both receptors were biotinylated at the AviTag site using the BirA Biotin Protein Ligase Reaction Kit (Avidity). Biotinylated receptors were captured with Series S CAP chips and Biotin CAPture reagent (Cytiva). Antibodies were titrated in running buffer (1x PBSP+, Cytiva) and pre-incubated with 10 nM NGF (TrkA assay) or 50 nM NGF (p75 assay) at the indicated ratios. Binding was assessed by injecting these samples over the captured receptor for 180 seconds. Rmax was used to calculate percent inhibition by dividing the Rmax of the premixed samples by the average Rmax of the NGF-only samples collected across the entire assay. The ability of each antibody to block NGF binding to feline TrkA and p75 is shown in Table 16. [Table 16]

[0151] Example 7 Testing the affinity maturation mutation G55R in canine clone SC-42_101_006

[0152] Feline clone 101 is a clone of canine clone SC-42_101_006 (V H Domain; SC-42_006V LThe affinity matured feline clone AHF17602 has been mutated to G55R to remove a potential NG deamidation site, which is also present in the canine clone. Clone SC-42_101_006 was mutated to R55 and both the parent and the R55 variant were transiently expressed in CHO cells and purified by Protein A. Variable domain sequences are shown in Figures 1 and 2. Affinity for NGF was assessed using SPR with a Biacore T200 instrument. Antibodies were captured using a Protein A Series S chip. NGF binding was then assessed at multiple concentrations starting at 50 nM using PBSP+running buffer (Cytiva) at a flow rate of 30 μL / min. Binding times were 120 s long and dissociation times were run at 600 s. The chip surface was regenerated with 10 mM glycine. Reference subtracted sensorgrams were fitted to a 1:1 binding model using the Biacore T200 Evaluation software. The data is shown in Table 17 below. [Table 17]

[0153] The invention is further described by the following numbered paragraphs:

[0154] 1. An isolated protein that specifically binds to canine NGF, (a) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 146), wherein X1 comprises A, G, or N, X2 comprises L or M, X3 comprises A, D, E, or S, X4 comprises F, I, L, M, or V, X5 comprises N or T, X6 comprises E, S, or T, X7 comprises G, H, N, S, or Q, and X8 comprises A or S; (b) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence X1X2SNGGT (SEQ ID NO: 147), wherein X1 comprises I or L and X2 comprises W or Y; (c) Amino acid sequence AX2IX4X5YX7X8X9YLX 12 X 13 YX 15 X 16 X 17 (SEQ ID NO: 148), wherein X2 comprises D, E, K, N, Q, S, or T, X4 comprises W or Y, X5 comprises F, H, W, or Y, X7 comprises D or E, X8 comprises A or S, X9 comprises D or Y, and X 12 contains H or Y, and X 13 contains F or W and X 15 contains F, I, L, W, or Y, and X 16 contains D or Q and X 17 comprises F, I, L, M, W, or Y; (d) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence X1X2IX4X5X6 (SEQ ID NO: 149), wherein X1 comprises D, E, or K, X2 comprises A, G, or N, X4 comprises G, N, Q, or S, X5 comprises N or S, and X6 comprises A, G, N, S, or T; (e) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence AX2X3 (SEQ ID NO: 150), wherein X2 comprises A, S, or T, and X3 comprises A, D, E, N, Q, S, or T; and (f) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence QX2GX4X5X6PX8T (SEQ ID NO: 151), wherein X2 comprises H or Q, X4 comprises F, H, W, or Y, X5 comprises K or Q, X6 comprises F or W, and X8 comprises L or M. The isolated protein comprising an antigen-binding portion comprising:

[0155] 2. (a) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence X1X2X3X4TX6X7S (SEQ ID NO: 152), wherein X1 comprises A or G, X2 comprises L or M, X3 comprises E or S, X4 comprises F or L, X6 comprises S or T, and X7 comprises H, N, or S; (b) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence IWSNGGT (SEQ ID NO: 153); (c) Amino acid sequence AX2IYYYX7ADYLHX 13 YX 15 DX 17 (SEQ ID NO: 154), wherein X2 comprises N, Q, or S; X7 comprises D or E; and X 13 contains F or W and X 15 contains F, I, L, W, or Y, and X 17 comprises F, I, L, or M; (d) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence X1GIX4NX6 (SEQ ID NO: 155), wherein X1 comprises D or E, X4 comprises Q or S, and X6 comprises G, N, S, or T; (e) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence ATX3 (SEQ ID NO: 156), wherein X3 comprises D, E, N, Q, or S; and (f) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence QQGX4X5X6PLT (SEQ ID NO: 157), wherein X4 comprises F, H, or Y, X5 comprises K or Q, and X6 comprises F or W. 2. The protein of paragraph 1, comprising an antigen-binding portion comprising:

[0156] 3. The protein of paragraph 1 or 2, comprising no more than two changes per VH-CDR compared to SEQ ID NO: 137 and no more than two changes per VL-CDR compared to SEQ ID NO: 138.

[0157] 4. The protein of paragraph 1 or 2, comprising no more than one change per VH-CDR compared to SEQ ID NO: 137 and no more than one change per VL-CDR compared to SEQ ID NO: 138.

[0158] 5. The protein of any one of paragraphs 1-4, comprising a heavy chain framework (FR1H+FR2H+FR3H+FR4H) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, or SEQ ID NO:141.

[0159] 6. The protein of any one of paragraphs 1-5, comprising a light chain framework (FR1L+FR2L+FR3L+FR4L) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:142.

[0160] 7. V comprising SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, or SEQ ID NO:141 H 7. The protein of any one of paragraphs 1 to 6, comprising a domain.

[0161] 8. V comprising SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:142 L8. The protein of any one of paragraphs 1 to 7, comprising a domain.

[0162] 9. An isolated nucleic acid sequence encoding an anti-NGF antibody or antibody fragment according to any one of paragraphs 1 to 8.

[0163] 10. A vector comprising the nucleic acid described in paragraph 9.

[0164] 11. A recombinant cell comprising a nucleic acid according to any one of paragraphs 9 or 10.

[0165] 12. A cell expressing a protein according to any one of paragraphs 1, a therapeutically effective amount of an anti-NGF protein according to any one of paragraphs 1 to 8, or a nucleic acid according to paragraph 9 or 10.

[0166] 13. A method for producing an anti-NGF protein according to any one of paragraphs 1 to 8, comprising culturing a host cell according to paragraph 11 under conditions which result in the production of said anti-NGF protein.

[0167] 14. A pharmaceutical composition comprising a therapeutically effective amount of an anti-NGF protein according to any one of paragraphs 1 to 8.

[0168] 15. A method for treating pain in a subject, comprising administering to said subject a therapeutically effective amount of an anti-NGF protein according to any one of paragraphs 1 to 8.

[0169] 16. The method of paragraph 15, wherein said pain comprises inflammatory pain, post-operative incision pain, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain due to burns, pain from trauma, musculoskeletal pain, rheumatic pain, or osteoporotic pain.

[0170] 17. The method of paragraph 16, wherein the subject comprises a dog.

[0171] 18. The method of paragraph 16, wherein the subject comprises a cat.

[0172] 19. The method of paragraph 16, wherein the subject comprises a human.

[0173] 20. A method for detecting NGF in a sample, the method comprising incubating a sample containing NGF in the presence of an anti-NGF protein described in any one of paragraphs 1 to 8, and detecting the anti-NGF protein bound to NGF in the sample.

[0174] Although preferred embodiments of the invention have been described in detail, it should be understood that the invention defined by the above paragraphs is not limited to the specific details set forth in the above description, since many obvious variations thereof are possible without departing from the spirit or scope of the invention.

Claims

1. An antigen-binding protein that specifically binds to nerve growth factor (NGF), (a) amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 (SEQ ID NO: 146), wherein X 1 contains A, G, or N, and X 2 contains L or M, and X 3 contains A, D, E, or S, and X 4 includes F, I, L, M, or V, and X 5 contains N or T, and X 6 contains E, S, or T, and X 7 contains G, H, N, S, or Q; X 8 comprises A or S; (b) amino acid sequence X 1 X 2 SNX 5 GT (SEQ ID NO: 147), wherein X 1 contains I or L, and X 2 contains W or Y, and X 5 comprises G or R; (c) amino acid sequence AX 2 IX 4 X 5 YX 7 X 8 X 9 YLX 12 X 13 YX 15 X 16 X 17 (SEQ ID NO: 148), wherein X 2 contains D, E, K, N, Q, S, or T, and X 4 contains W or Y, and X 5 contains F, H, W, or Y, and X 7 contains D or E, and X 8 contains A or S, and X 9 contains D or Y, and X 12 contains H or Y, and X 13 contains F or W, and X 15 contains F, I, L, W, or Y, and X 16 contains D or Q, and X 17 the VH-CDR3 comprises F, I, L, M, W, or Y; (d) amino acid sequence X 1 X 2 IX 4 X 5 X 6 (SEQ ID NO: 149), wherein X 1 contains D, E, or K, and X 2 contains A, G, or N, and X 4 contains G, N, Q, or S, and X 5 contains N or S, and X 6 comprises A, G, N, S, or T; (e) amino acid sequence AX 2 X 3 (SEQ ID NO: 150), wherein X 2 contains A, S, or T, and X 3 comprises A, D, E, N, Q, S, or T; and (f) amino acid sequence QX 2 GX 4 X 5 X 6 PX 8 T (SEQ ID NO: 151), wherein X 2 contains H or Q, and X 4 contains F, H, W, or Y, and X 5 contains K or Q, and X 6 contains F or W, and X 8 comprises L or M The antigen-binding protein comprising:

2. (a) amino acid sequence X 1 X 2 X 3 X 4 TX 6 X 7 S (SEQ ID NO: 152), wherein X 1 contains A or G, and X 2 contains L or M, and X 3 contains E or S, and X 4 contains F or L, and X 6 contains S or T, and X 7 comprises H, N, or S; (b) Amino acid sequence IWSNX 5 GT (SEQ ID NO: 153), wherein X 5 comprises G or R; (c) amino acid sequence AX 2 IYYYX 7 ADYLHX 13 YX 15 DX 17 (SEQ ID NO: 154), wherein X 2 contains N, Q, or S, and X 7 contains D or E, and X 13 contains F or W, and X 15 contains F, I, L, W, or Y, and X 17 the VH-CDR3 comprises F, I, L, or M; (d) amino acid sequence X 1 GIX 4 NX 6 (SEQ ID NO: 155), wherein X 1 contains D or E, and X 4 contains Q or S, and X 6 comprises G, N, S, or T; (e) amino acid sequence ATX 3 (SEQ ID NO: 156), wherein X 3 comprises D, E, N, Q, or S; and (f) amino acid sequence QQGX 4 X 5 X 6 PLT (SEQ ID NO: 157), wherein X 4 contains F, H, or Y, and X 5 contains K or Q, and X 6 the VL-CDR3 comprises F or W 2. The antigen-binding protein of claim 1, comprising:

3. 2. The antigen binding protein of claim 1, which comprises no more than two substitutions per VH-CDR compared to SEQ ID NO: 137 and no more than two substitutions per VL-CDR compared to SEQ ID NO:

138.

4. 2. The antigen binding protein of claim 1, which comprises no more than one substitution per VH-CDR compared to SEQ ID NO: 137 and no more than one substitution per VL-CDR compared to SEQ ID NO:

138.

5. 2. The antigen binding protein of claim 1, comprising one or more VH-CDRs of any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, SEQ ID NO:141, or SEQ ID NO:207, and one or more VL-CDRs of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:

142.

6. 2. The antigen binding protein of claim 1, comprising the VH-CDRs of any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, SEQ ID NO:141, or SEQ ID NO:207, and the VL-CDRs of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:

142.

7. 2. The antigen-binding protein of claim 1, comprising the VH-CDRs and VL-CDRs of SC-42_006, SC-42_007, SC-42_008, SC-42_010, SC-42_011, SC-42_023, SC-42_032, SC-42_045, SC-42_047, SC-42_048, SC-42_052, SC-42_070, SC-42_073, SC-42_077, SC-42_082, SC-42_090, or SC-42_101.

8. 2. The antigen binding protein of claim 1, wherein the CDRs are according to the IMGT system.

9. 2. The antigen binding protein of claim 1, wherein the CDRs are in accordance with Kabat.

10. 2. The antigen binding protein of claim 1, wherein the CDRs are according to Chothia.

11. 2. The antigen binding protein of claim 1, comprising a heavy chain framework (FR1H+FR2H+FR3H+FR4H) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, SEQ ID NO:141, or SEQ ID NO:

207.

12. 2. The antigen binding protein of claim 1, comprising a light chain framework (FR1L+FR2L+FR3L+FR4L) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:

142.

13. a V that is at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, SEQ ID NO:141, or SEQ ID NO:207; H 2. The antigen-binding protein of claim 1, comprising a domain.

14. a V that is at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:142; L 2. The antigen-binding protein of claim 1, comprising a domain.

15. An antigen-binding protein that specifically binds to NGF, (a) amino acid sequence X 1 LX 3 X 4 X 5 X 6 X 7 X 8 MX 10 wherein X is a heavy chain complementarity determining region 1 (VH-CDR1) comprising: 1 contains A, G, L, N, or Q; X 3 includes A, D, E, G, H, I, M, S, T, or Y; and X 4 contains L, M, or V, and X 5 contains A, M, N, R, S, T, or V; X 6 contains A, E, G, H, K, R, S, or T; X 7 includes A, D, H, I, N, Q, S, T, or Y; and X 8 contains A or S, and X 10 comprises S or V; (b) amino acid sequence X 1 X 2 X 3 X 4 X 5 GTX 8 YX 10 DX 12 VX 14 a heavy chain complementarity determining region 2 (VH-CDR2) comprising: 1 contains I or L, and X 2 contains W or Y, and X 3 contains A, P, or S, and X 4 contains D, E, N, Q, R, or S; and X 5 contains G, R, or Y, and X 8 contains D or Y, and X 10 contains D, E, H, S, or T, and X 12 contains D or S, and X 14 comprises D, E, or K; (c) amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 LX 12 X 13 X 14 FX 16 X 17 a heavy chain complementarity determining region 3 (VH-CDR3) comprising: 1 contains A, D, E, K, N, Q, S, or T, and X 2 includes A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; and X 3 contains I, L, W, or Y, and X 4 contains F, T, W, or Y, and X 5 contains F, H, or Y, and X 6 contains H or Y, and X 7 contains D or E, and X 8 contains A, S, or V, and X 9 includes D, E, H, K, N, Q, or Y; X 10 contains F, H, or Y, and X 12 contains H or Y, and X 13 contains F or W, and X 14 includes D, I, L, W, or Y, and X 16 contains D or Q, and X 17 the VH-CDR3 comprises E, F, H, I, L, M, N, P, W, or Y; (d) amino acid sequence X 1 ASX 4 X 5 X 6 X 7 X 8 X 9 LX 11 a light chain complementarity determining region 1 (VL-CDR1) comprising: 1 contains F or R, and X 4 contains E, K, or N, and X 5 contains A or G, and X 6 contains I, L, or V, and X 7 includes A, D, G, L, P, Q, S, V, or Y; and X 8 contains K, Q, N, S, or Y, and X 9 includes A, D, E, F, G, H, K, L, N, Q, R, S, or T; and X 11 comprises A, G, or S; (e) amino acid sequence AX 2 X 3 X 4 X 5 X 6 X 7 a light chain complementarity determining region 2 (VL-CDR2) comprising: 2 includes A, D, L, Q, S, T, V, or Y; and X 3 contains D, E, K, N, Q, or S, and X 4 comprises H, I, K, L, M, N, or V; X 5 contains H or L, and X 6 contains H, I, L, or M, and X 7 comprises D, E, N, S, or T; (f) amino acid sequence QQX 3 X 4 X 5 X 6 X 7 X 8 a light chain complementarity determining region 3 (VL-CDR3) comprising T, wherein X 3 contains G or Y, and X 4 includes D, F, G, H, K, L, R, S, T, V, W, or Y; and X 5 contains E, K, Q, R, or S, and X 6 contains I, F, T, or W, and X 7 contains E or P, and X 8 comprises L, M, or W. The antigen-binding protein comprising:

16. VH-CDR1 is GLSLTSX 7 SMX 10 wherein X 7 contains A, D, N, or S, and X 10 contains S or V, VH-CDR2 is X 1 X 2 SNX 5 GT, wherein X 1 contains I or L, and X 2 contains W or Y, and X 5 contains G or R, VH-CDR3 is ASIYYYX 7 AX 9 YLHWYFDX 17, wherein X 7 contains D or E, and X 9 contains D or E, X 17 contains E or F, VL-CDR1 is RASX 4 GIX 7 X 8 NLS, wherein X 4 contains E or K, and X 7 contains A, Q, or S, and X 8 contains K or N, VL-CDR2 is AX 2 X 3 X 4 LHS, wherein X 2 contains Q or T, and X 3 contains D or S, and X 4 comprises I, N, or V; VL-CDR3 is QQGX 4 KWPLT, wherein X 4 comprises F, W, or Y.

17. 16. The antigen binding protein of claim 15, which comprises no more than two substitutions per VH-CDR compared to SEQ ID NO: 204 and no more than two substitutions per VL-CDR compared to SEQ ID NO:

199.

18. 16. The antigen binding protein of claim 15, which comprises no more than one substitution per VH-CDR compared to SEQ ID NO: 204 and no more than one substitution per VL-CDR compared to SEQ ID NO:

199.

19. 16. The antigen binding protein of claim 15, comprising one or more VH-CDRs of any one of SEQ ID NO:141, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, or SEQ ID NO:206, and one or more VL-CDRs of any one of SEQ ID NO:142, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, or SEQ ID NO:

203.

20. 16. The antigen binding protein of claim 15, comprising the VH-CDRs of any one of SEQ ID NO:141, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, or SEQ ID NO:206, and the VL-CDRs of any one of SEQ ID NO:142, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, or SEQ ID NO:

203.

21. 16. The antigen binding protein of claim 15, comprising the VH-CDRs and VL-CDRs of clone AHF17591, AHF17592, AHF17593, AHF17594, AHF17595, AHF17596, AHF17597, AHF17602, AHF17603, AHF17607, AHF17609, SC-184_76, SC-184_102, SC-184_110, SC-184_76-Arg, SC-184_102-Arg, or SC-184_110-Arg.

22. 16. The antigen binding protein of claim 15, comprising a heavy chain framework (FR1H+FR2H+FR3H+FR4H) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:141, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, or SEQ ID NO:

206.

23. 16. The protein of claim 15, comprising a light chain framework (FR1L+FR2L+FR3L+FR4L) that is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:142, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, or SEQ ID NO:

203.

24. V that is at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:141, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, or SEQ ID NO:

206. H 16. The antigen binding protein of claim 15, comprising a domain.

25. 16. The antigen binding protein of claim 15, comprising a VL domain that is at least 80%, or at least 85%, or at least 90%, or at least 93%, or at least 95% identical to SEQ ID NO:142, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, or SEQ ID NO:

203.

26. 26. An isolated nucleic acid having a nucleic acid sequence encoding the antigen-binding protein or antibody fragment thereof of any one of claims 1 to 25.

27. A vector comprising the nucleic acid of claim 26.

28. 27. A recombinant cell comprising the nucleic acid of claim 26.

29. A cell expressing the antigen-binding protein of any one of claims 1 to 25.

30. A method of producing an antigen-binding protein according to any one of claims 1 to 25, comprising culturing cells expressing the antigen-binding protein under conditions which result in the production of said antigen-binding protein according to any one of claims 1 to 25.

31. 26. A pharmaceutical composition comprising a therapeutically effective amount of the antigen-binding protein of any one of claims 1 to 25.

32. 32. The pharmaceutical composition of claim 31 for the treatment or alleviation of pain in a subject.

33. 33. The pharmaceutical composition of claim 32, wherein the pain comprises inflammatory pain, post-operative incision pain, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain due to burns, pain from trauma, musculoskeletal pain, rheumatic pain, or osteoporotic pain.

34. 34. The pharmaceutical composition of claim 33, wherein the subject comprises a dog.

35. 34. The pharmaceutical composition of claim 33, wherein the subject comprises a cat.

36. 34. The pharmaceutical composition of claim 33, wherein the subject comprises a human.

37. 26. A method for detecting NGF in a sample, the method comprising incubating a sample containing NGF in the presence of the antigen-binding protein of any one of claims 1 to 25, and detecting the antigen-binding protein bound to NGF in the sample.

Images