TW201321406A - Tumour necrosis factor antibodies and methods of using the same - Google Patents

Abstract

Caninised and chimeric antibodies and antigen binding fragments thereof which bind specifically to canine tumour necrosis factor and inhibit the ability of canine TNF to bind to the TNFR1 receptor are provided. The invention further extends to nucleic acids encoding same and to methods of treating chronic inflammatory disease such as arthritis in a canine using said antibodies and/or nucleic acids.

Description

Tumor necrosis factor antibody and method of using same

The present invention relates to a binding antibody and a fragment thereof which act as inhibitors of canine tumor necrosis factor alpha. The invention extends to methods of making the same and the therapeutic use of such antibodies and fragments in the treatment of canine necrosis factor mediated chronic inflammatory conditions, such as arthritis.

Tumor necrosis factor alpha (TNF-α, TNF) is a potent cytokine that plays a multi-functional role in immunity, inflammation, control of cell proliferation, differentiation and apoptosis. Inhibition of TNF using tumor necrosis factor receptor (TNFR)-immunoglobulin Fc domain fusion protein or anti-TNF neutralizing monoclonal antibody has been demonstrated to be a successful treatment for a variety of human inflammatory diseases including rheumatoid arthritis and psoriasis joints. The treatment of inflammation).

Companion animals (such as dogs) develop inflammatory conditions similar to osteoarthritis, immune-mediated polyarthritis, plasma-lymphocytitis, systemic lupus erythematosus (SLE), vasculitis, and various autoimmune skins. disease. It is estimated that one in five adult dogs in the United States has arthritis, and dogs have been used as models for human joint diseases such as osteoarthritis, anterior cruciate ligament rupture, and meniscus injury.

The role of TNF in the development of inflammation in dogs has been well documented. For example, in a closed-chest model, treatment of dogs with TNF inhibitor etanercept (huTNFR-Fc) reduced myocardial damage by approximately 25% to 40 after ischemia-reperfusion induced by balloon occlusion. %, with a decrease in associated inflammatory markers such as ICAM-1 and NF-kB. Similarly, it has also been shown The use of 2 mg/kg TNFR-Fc in the open-chest model of ischemia-reperfusion reduced the infarct size by 60%.

In addition, the secretion of TNFα is increased in the dog synovial cell infiltrate presented with knee arthritis. TNF and type II TNF receptors have been shown to be significantly elevated in the central and peripheral retina of glaucoma dogs as part of a broad inflammatory response. Anti-canine TNF MAb has been used to detect low levels of TNF by capture ELISA in supernatants of LPS-treated canine PBMC. The performance of TNFα in skin samples of canine angioderma, hair blastoma, lipoma and obese cell tumor has also been reported. In the induced osteoarthritis model, TNF-α and TNF receptors are present in canine articular cartilage. The humanized monoclonal antibody adalimumab against TNF has been tested in two dogs with exfoliative cutaneous lupus erythematosus (ECLE) (0.5 mg/kg every 2 weeks for 8 weeks), but The disease progression was unchanged and there was no change in serum TNF-α content.

Therefore, since TNF is involved in numerous inflammation-mediated conditions in dogs, canine TNF inhibitors are needed, which can be used for long-term inhibition of TNF to prevent or treat such chronic inflammatory conditions.

The inventors have surprisingly identified a method of preparing antibodies that, upon extensive work, produces antibodies and binding fragments that specifically bind to canine TNF and neutralize the biological activity of canine TNF. In particular, it is shown herein that, although many amino acid modifications are introduced into the framework regions of antibodies, the antibodies and binding fragments of the invention bind to canine TNF with higher specificity due to the novel and inventive manner of performing such modifications. . It is further shown that the biological activity of canine TNF is chelated by inhibiting the binding of canine TNF to the TNF receptor expressed by the cell membrane. This in turn will prevent the development or progression of TNF-mediated induction, a disease mediated by canine inflammation, such as arthritis.

The anti-system of the invention is produced using recombinant DNA methods and exhibits binding specificity for canine TNF, as well as canine framework and constant domain sequences to reduce immunogenicity when administered to a canine host. Therefore, the risk of xenogeneic antibody induction is minimized. Since antibodies are not produced using standard methods, such as CDR grafting or the like, their binding specificity to canine TNF is completely surprising and unexpected.

According to a first aspect of the invention, a caninised antibody or antigen-binding fragment thereof which specifically binds to canine tumor necrosis factor (TNF) is provided.

In certain embodiments, the anti-system is prepared by a process comprising or consisting essentially of: providing a donor antibody from a species other than a canine, wherein the donor antibody has binding specificity for tumor necrosis factor - each amino acid residue of the amino acid sequence of the framework region of the donor antibody and the corresponding amino acid residue at the corresponding position in the amino acid sequence of the framework region of one or more canine antibodies Bases are compared to identify one or more amino acid residues at the corresponding positions in the amino acid sequence of the framework region of one or more canine antibodies in the amino acid sequence of the framework region of the donor antibody One or more amino acid residues; and - replacing one or more identified amines in the donor antibody with one or more amino acid residues present at corresponding positions in one or more canine antibodies Base acid residue.

The above methods modify the donor antibody in a manner for use in dogs in such a way that the modified antibody does not contain any amino acid that will be foreign in the canine position at any position within the framework region. Thus, a modified antibody retains the specificity and affinity of the donor antibody for the antigen of interest, but it is important that it is modified such that no potentially foreign epitopes are produced. Therefore, the modified antibody is not considered to be foreign in dogs, and thus does not induce an immune response in the dog that can cause neutralizing of the efficacy of the antibody, especially after long-term administration.

In certain embodiments, the step of substituting one or more identified amino acid residues comprises replacing one or more identified amino acids with one or more amino acid residues present at the respective positions. A residue having the highest homology to one or more substituted amino acid residues.

In certain embodiments, the method further comprises the step of replacing the constant domain of the heavy and/or light chain of the donor antibody with a constant domain derived from a heavy chain and/or a light chain of a canine antibody.

The above method does not include CDR grafting. The antibody prepared according to the above method comprises the CDR of the donor antibody, the canineized framework region prepared according to the above method, and the canine constant domain.

The invention extends to antibodies prepared according to the above methods, such as the antibodies described below, and fragments thereof.

Thus, according to another aspect of the present invention, there is provided a composition comprising a peptide or a nucleic acid sequence encoding a peptide, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. The peptide typically binds to tumor necrosis factor alpha. In certain embodiments, the peptide is a canineized antibody or a chimeric antibody or a binding fragment thereof. In certain embodiments, canineized antibodies are prepared according to the methods described above for the preparation of antibodies.

In certain aspects, the invention provides a composition comprising a canine tumor necrosis factor alpha binding agent or an oligonucleotide encoding a tumor necrosis factor alpha binding agent, wherein the binding agent comprises an amino acid selected from the group consisting of Sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: SEQ ID NO:9.

Another aspect of the invention provides a canineized antibody or chimeric antibody or antigen-binding fragment thereof that specifically binds to canine tumor necrosis factor (TNF), in particular canine TNF[alpha] (TNF-[alpha]). Canineized antibodies or chimeric antibodies or antigen-binding fragments derived therefrom typically neutralize the biological activity of canine TNF. In particular, the ability of canineized antibodies or chimeric antibodies or binding fragments to bind to canine TNF to chelate canine TNF binding and activate membrane-associated canine TNF receptors. In certain embodiments, the canineized antibody or chimeric antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. In certain embodiments, canineized antibodies are prepared according to the methods described above for the preparation of antibodies.

Another aspect of the invention provides a canineized antibody that binds to complement and initiates complement-mediated TNF-[alpha] expression of cytolysis. These antibodies will reduce the number of inflammatory cells that TNF exhibits and thus produce long-term resistance in vivo. Inflammatory activity. In certain embodiments, canineized antibodies are prepared according to the methods described above for the preparation of antibodies.

In certain aspects of the above-described specific examples of the present invention, a canine chimeric antibody or humanized antibody or binding fragment, or less than 1 × 10 ^-8 1 × 10 ^-8 K _D of binding affinity to TNF.

In another or related aspect of the invention, a neutralizing antibody or antigen-binding fragment thereof is capable of specifically binding to canine tumor necrosis factor alpha (TNF-alpha), the antibody or antibody binding fragment comprising the following , consisting of or consisting essentially of: an amino acid sequence comprising SEQ ID NO: 1 or at least 85%, 90%, 95% or 99 with the amino acid sequence of SEQ ID NO: 1. The light chain variable domain of the % consensus amino acid sequence, and/or comprising the amino acid sequence of SEQ ID NO: 2 or at least 85%, 90%, 95 with the amino acid sequence of SEQ ID NO: 2. The heavy chain variable region of the amino acid sequence of % or 99% identity. The antibody is typically a chimeric or canine antibody.

In certain embodiments in which the antibody is a canineized antibody or fragment thereof, it comprises, consists of, or consists essentially of: an amino acid sequence comprising SEQ ID NO: 3 or with SEQ ID NO: The light chain of the amino acid sequence of 3 having at least 85%, 90%, 95% or 99% identity of the amino acid sequence.

In certain embodiments, the heavy chain variable region (VH) binds to another amino acid sequence comprising at least one immunoglobulin constant domain. In certain embodiments, the immunoglobulin constant domain is derived from an antibody to a subclass of IgG (immunoglobulin G) to form the entire heavy chain of a canineized antibody of the invention. Four different canine heavy chain constant domains are known. The constant domains typically comprise CH1, CH2 and The CH3 region and a suitable linker (or "hinge") between the CH1 and CH2 regions.

In certain embodiments, a canineized antibody or antibody binding fragment comprises, consists of, or consists essentially of: comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID Amino acid sequence of the group consisting of NO: 6 and SEQ ID NO: 7 or a heavy chain of an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to the amino acid sequence. In certain embodiments, a chimeric antibody or antibody binding fragment comprises, consists of, or consists essentially of: an amino acid comprising SEQ ID NO: 9 or with SEQ ID NO: 9 The amino acid has a heavy chain of amino acid sequences having a sequence identity of at least 85%, 90%, 95% or 99%. In certain embodiments, a chimeric antibody comprises an amino acid sequence having a population selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22 or at least 85% with the amino acid sequence The heavy chain of the amino acid sequence of 90% or 95% sequence homology.

In a specific example where the antibody is a canineized antibody, the antibody has an amino acid sequence in which the residue has been altered by deletion, addition and/or substitution to deimmunize the sequence such that it does not A heterologous antibody against the antibody is produced. In particular, antibodies comprise a framework derived from a canine and a constant domain amino acid sequence.

In another aspect, the invention extends to a neutralizing antibody or antigen-binding fragment thereof that specifically binds to canine tumor necrosis factor (TNF), the antibody or antibody binding fragment comprising a light chain and a heavy chain, and a light chain And the heavy chain consists of or consists essentially of a light chain and a heavy chain, wherein the light chain variable region (VL) comprises SEQ ID NO: an amino acid sequence of 1 or an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, and wherein the heavy chain variable region (VH) comprises, consists of, or consists essentially of: an amino acid sequence identical or substantially homologous to the amino acid sequence of SEQ ID NO: 2, or with the amino group The acid sequence has an amino acid sequence of at least 85%, 90%, 95% or 98% sequence identity.

In certain embodiments, the antibody is a canineized antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 3, consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: ID NO: amino acid sequence composition of 3; and/or a heavy chain comprising, consisting of, or consisting essentially of: selected from the group consisting of SEQ ID NO: 4, SEQ ID NO : 5, the amino acid sequence of the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7 or having at least 85%, more preferably 95% amino acid identity and preferably at least 98% identical to the amino acid sequence Sequence of sex. The light chain is typically a kappa light chain.

In certain embodiments, the antibody is a chimeric antibody comprising a light chain comprising, consisting of, or consisting essentially of: the amino acid sequence of SEQ ID NO: Or a sequence having at least 85%, more preferably 95% amino acid identity and preferably at least 98% identity with the amino acid sequence of SEQ ID NO: 8; and/or a heavy chain comprising the following , consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: 9 or having at least 85%, more preferably 95% amino acid, with the amino acid sequence of SEQ ID NO: Sexual and optimal sequence of at least 98% identity. The light chain is typically a kappa light chain.

In other embodiments, the antibody or binding fragment of the invention is extended to An aglycosylated variant in which a constant domain amino acid residue suitable for glycosylation is deleted or substituted with a residue that is not glycosylated to prevent glycosylation.

The inventors have additionally defined a series of framework regions (FR) that can be combined with complementarity determining regions (CDRs) to form canineized heavy and light chain variable domains. The heavy and light chain domains each have four framework regions, designated FR1, FR2, FR3, and FR4.

The antibody molecule can comprise a heavy chain variable domain comprising the CDR1, CDR2 and CDR3 regions and associated insertion framework regions. The heavy chain variable domain (VH) CDRs are referred to as VHCDRs, wherein such CDRs are found at the following positions according to the Kabat numbering system: VHCDR1-Kabat residues 31-35, VHCDR2-Kabat residues 50-65, VHCDR3-Kabat residues 95-102 (Kabat EA et al. (1991) Sequences of proteins of immunological interest, 5th ed. Bethesda: US Department of Health and Human Services). Furthermore, the antibody further comprises a light chain variable domain comprising the CDR1, CDR2 and CDR3 regions and associated insertion framework regions. The light chain variable domain (VL) CDRs are referred to as VLCDRs, wherein such CDRs are found at the following amino acid residue positions according to the Kabat numbering system: VLCDR1-Kabat residues 24-34, VLCDR2-Kabat residues 50-56, VLCDR3-Kabat residue 89-97. The light or heavy chain variable domain comprises four framework regions FR1, FR2, FR3 and FR4, which are inserted by CDRs in the following arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In another aspect, the invention extends to an anti-canine TNF antibody or a canine TNF binding fragment thereof, the antibody or antibody binding fragment comprising a light chain variable region comprising at least one of: The FR1 framework region consists of or comprises the amino acid sequence of SEQ ID NO: 10, the FR2 framework region, consists of the amino acid sequence of SEQ ID NO: 11 or comprises SEQ ID NO Amino acid sequence of 11 , FR3 framework region, consisting of or comprising the amino acid sequence of SEQ ID NO: 12, and the FR4 framework region, the amine of SEQ ID NO: 13. The acid sequence comprises or comprises the amino acid sequence of SEQ ID NO: 13; and/or the heavy chain variable region comprising at least one of: an FR1 framework region, an amine of SEQ ID NO: The acid sequence consists of or comprises the amino acid sequence of SEQ ID NO: 14, the FR2 framework region, consists of the amino acid sequence of SEQ ID NO: 15 or comprises the amino acid sequence of SEQ ID NO: 15, the FR3 framework region, The amino acid sequence consisting of or comprising the amino acid sequence of SEQ ID NO: 16, and the FR4 framework region, consisting of or comprising the amino acid sequence of SEQ ID NO: 17. Amino acid sequence.

The light and heavy chain CDRs are typically derived from antibodies that have binding specificity for canine TNF.

The canineized anti-canine TNF antibodies of the invention are typically produced by recombinant DNA processes that do not require the introduction of back mutations into the framework regions of the light or heavy chain variable domains.

In certain embodiments, a light chain variable domain comprising the at least one framework region described above binds to a canine light chain constant domain, typically a light chain κ Constant domain, but λ light chain exists as appropriate. In certain embodiments, the light chain comprises an FR1 region having the amino acid sequence of SEQ ID NO: 10, an FR2 region having the amino acid sequence of SEQ ID NO: 11, and an amine group having SEQ ID NO: The FR3 region of the acid sequence and the FR4 region having the amino acid sequence of SEQ ID NO: 13, or having at least 60%, 70%, 80%, 90%, 95% or 98% sequence identity to the amino acid sequence described above The framework region of the amino acid sequence. In some embodiments, the identity is over the length of at least about 5 amino acids, preferably about 10 amino acids.

In certain other specific embodiments, the heavy chain variable region comprising at least one of the framework regions described above binds to a heavy chain constant domain derived from a canine. In certain embodiments, the constant domain amino acid sequence lacks any post-translational modifications, or can be modified to remove any or all residues that can undergo N-linked or O-linked glycosylation to achieve a constant domain No glycosylation. In certain embodiments, the heavy chain comprises an FR1 region having the amino acid sequence of SEQ ID NO: 14, an FR2 region having the amino acid sequence of SEQ ID NO: 15, and an amino acid having SEQ ID NO: The FR3 region of the sequence and the FR4 region having the amino acid sequence of SEQ ID NO: 17 or having at least 60%, 70%, 80%, 90%, 95% or 98% sequence identity to the amino acid sequence described above The framework region of the amino acid sequence. In some embodiments, the identity is over the length of at least about 5 amino acids, preferably about 10 amino acids.

In some other specific examples, the framework regions described herein can be modified. That is, the inventors have identified that for some of the residues in each framework region, an amino acid residue which may be present at a predetermined position is selected. Importantly, these framework region modifications do not cause a conformational change in the complementarity determining region, as this Configurational changes may adversely alter the binding specificity and/or affinity of the resulting antibody. In certain embodiments, the invention extends to the introduction of two or more amino acid substitutions into the amino acid residues of the framework regions of the light chain variable region and/or the heavy chain variable region.

Thus, in certain other specific embodiments, the invention extends to a polypeptide, such as an antibody or antigen-binding fragment thereof, comprising a light chain variable domain having an FR1 region comprising one or more of the following amino acids Substituting the modified amino acid sequence of SEQ ID NO: 10 (wherein the amino acid is represented by its one-letter code): the amino acid residue T (T5) at position 5 is replaced by an amino acid residue M or I; S7 is T; A9 is L or P; S12 is A; L13 is V; S14 is T or R; Q15 is P or R; E16 is D; K18 is E, A, P, T or L; V19 is A; T20 is S; T22 is S or Y; and C23 is Y.

In certain other embodiments, the light chain FR2 region having the amino acid sequence of SEQ ID NO: 11 can be modified with one or more of the following amino acid substitutions: Y2 is F, I or L, and Q3 is R, L. Or I, Q4 is H, K5 is R, P6 is S or A, G7 is D, A9 is S, T or P, K11 is Q, E or R, and L12 is R, P, G, A or S, I14 Is L, and Y15 is F, N, S, E or V.

In certain other embodiments, the light chain FR3 region having the amino acid sequence of SEQ ID NO: 12 can be modified with one or more of the following amino acid substitutions: G1 is A, V2 is A, and P3 is S, S4. Is D, F6 is L or V, S7 is I, G8 is A, T13 is A, D14 is E, T16 is S or R, L17 is F, T18 is R or K, and S21 is R, G or T, L22 Is V, P24 is A, E25 is D, G, I or N, V27 is A, T, G or S, A28 is G, and V29 Is I or L.

In certain other embodiments, the light chain FR4 region having the amino acid sequence of SEQ ID NO: 13 can be modified with one or more of the following amino acid substitutions: G2 is S, Q3 is A, P or T, G4 Is E, T5 is P, K6 is Q or S, V7 is L or W, E8 is D or R, and I9 is L.

In certain other specific embodiments, the heavy chain FR1 region having the amino acid sequence of SEQ ID NO: 14 can be modified with one or more of the following amino acid substitutions: E1 is D or G, and V2 is G, L, E. , I or M, Q3 is H, R, A, V, E, K, L, P or S, L4 is V or P, V5 is A, L, E or M, E6 is Q or A, and S7 is F , L or T, G9 is E, G10 is D, A, N, E or T, L11 is Q, R, V or W, V12 is A, I or M, Q13 is K, R or N, and P14 is F Or T, G15 is A, E or T, G16 is E or A, S17 is T or P, L18 is R, R19 is K, T, G or V, L20 is I or V, S21 is Y, A23 is V , L, I or E, A24 is T, V, G, I or S, S25 is P, G or T, G26 is D, R or T, and F27 is L, I, S, D, T or V.

In certain other specific embodiments, the heavy chain FR2 region having the amino acid sequence of SEQ ID NO: 15 can be modified with one or more of the following amino acid substitutions: W1 is C and V2 is I, A, F or L. Q4 is L or H, A5 is S, T, G, P, V or D, P6 is L, G7 is E, R or L, K8 is R, E, G, A, M or Q, and G9 is E , R, D, T or V, L10 is T, P, F or M, E11 is Q, H, D, L, P or R, W12 is L, C, S, Y, F or M, and V13 is L , I or F, and S14 is A, T, G or L.

In certain other specific embodiments, the amino acid of SEQ ID NO: 16 The heavy chain FR3 region of the sequence may be modified by one or more of the following amino acid substitutions: R1 is Q, F2 is V or L, T3 is A, I or S, 14 is V, L, M or T, and S5 is A. , F or T, R6 is K, D7 is E or N, N8 is D, T, S, I or G, A9 is G, V, S, D, P or T, and K10 is R, E, N, Q , G or M, N11 is D, S, K, H or R, S12 is T, M, I or A, L13 is V, M, A or I, Y14 is F, H, S or T, L15 is I , Q16 is H, E, D, R or A, M17 is L, N18 is D, S, T, H, K, P or R, S19 is G, D, R, N or T, L20 is V, R21 Is T, G, K, S or I, A22 is V, D, T, S, G or P, E23 is D, A or V, T25 is A, S or M, A26 is G or V, and V27 is M , I, L, F, T, K or Q, Y28 is H, Y29 is F or H, A31 is V, T, G, M, R, S, C or L, and K32 is R, S, N, G, A, T, P, D, Q, V, E, I, M.

In certain other specific embodiments, the heavy chain FR4 region having the amino acid sequence of SEQ ID NO: 17 can be modified with one or more of the following amino acid substitutions: W1 is L, G2 is A or S, and Q3 is P. , H, R or D, T5 is A, S, I or N, L6 is S, Q, P or R, V7 is L, I or P, and T8 is F, I, A, S, L, P or Y , V9 is A, S10 is A, C, P or T, and S11 is L, A or P.

In certain embodiments, the antibody is a monoclonal antibody. The antibody is typically a canineized antibody or fragment thereof. Alternatively, the antibody is a chimeric antibody or a fragment thereof.

In certain other specific embodiments, the canineized or chimeric TNF-neutralizing antibody of the invention or a binding fragment derived therefrom has an equilibrium dissociation constant (K _D ) of 1×10 ^-8 or less than 1×10 ^-8 Binding affinity specifically binds to canine TNF-α (tumor necrosis factor alpha). Furthermore, it is preferred that the canineized antibody or chimeric antibody does not cross-react with any other epitope present in the dog, and additionally does not produce a heterologous antibody against the antibody of the present invention when administered to a dog. Furthermore, it is preferred that the constant domain of canine TNF-binding antibodies does not mediate any downstream effector functions, including (but not limited to) complement fixation and activation, ADCC and Fc receptor binding, when short-term anti-inflammatory activity is not required in vivo. And activation. The constant domains are selected from the group consisting of SEQ ID NO: 4, aglycosylated SEQ ID NO: 4, aglycosylated SEQ ID NO: 5, aglycosylated SEQ ID NO: 6, sugar free SEQ ID NO: 7 and SEQ ID NO: 7 were ligated.

In certain other embodiments, it is preferred that the constant domain of canine TNF-binding antibodies mediates, including but not limited to, complement fixation and activation, ADCC and Fc receptor binding and activation, when long-term anti-inflammatory activity is desired. The effector function is to eliminate canine TNF-expressing cells in vivo. The constant domains are selected from the heavy chains consisting of SEQ ID NO: 5 and SEQ ID NO: 6.

In certain other embodiments, the antibodies of the invention can be subjected to modification of the amino acid sequence of the heavy chain constant region. Such modifications may involve the addition, substitution or deletion of one or more amino acid residues. These amino acid changes are typically made to alter the functional characteristics of the antibody. For example, amino acid modifications can be made to block downstream effector functions mediated by antibody constant domains, such as by blocking the ability of an antibody to bind to an Fc receptor, activate complement, or induce ADCC. In addition, amino acid residues of the heavy chain constant domain can be modified to alter the circulating half-life of the antibody when administered to a dog.

The invention extends to antibody fragments that bind to canine TNF and chelate the ability of canine TNF to bind to TNFR.

In certain embodiments, an antibody binding fragment can comprise a heavy chain and a light chain sequence of the invention, the heavy and light chain sequences joined by a flexible linker to form a single chain antibody.

Single-chain Fv (scFv) contains the VH and VL domains. VH associates with the VL domain to form a target binding site. These two domains are covalently linked by a peptide linker. The scFv molecule may have a VL-linker-VH form if the N-terminus requires a light chain variable domain, or a VH-linker-VL form if the N-term requires a VH domain. Thus, in certain other specific examples, the antigen-binding fragment is a single-chain Fv (scFv) antibody fragment. In certain other specific embodiments, the antibody binding fragment is selected from the group consisting of, but not limited to, Fab antibody fragments, Fab' antibody fragments, F(ab') ₂ antibody fragments, Fv antibody fragments, scFV antibody fragments, and It is similar to the fragment.

In some embodiments, the invention provides multispecific or multivalent antibodies comprising an anti-TNF antibody or binding fragment of the invention that is coupled or conjugated to other antibodies having different binding specificities for use in combination therapy. The multispecific antibody comprises at least one antibody or binding fragment specific for the first TNF epitope, and at least one binding site specific for the other epitope present on the TNF or specific for the different antigen. Multivalent antibodies comprise antibodies or antibody binding fragments that have binding specificity for the same TNF epitope. Thus, in certain embodiments, the invention extends to an antibody fusion protein comprising four or more Fv regions or Fab regions of a canineized antibody of the invention. Another embodiment extends to an antibody fusion protein comprising one or more Fab or Fv regions derived from one or more Fab regions of an antibody described herein and from an antibody specific for TNF. In a certain In some other specific embodiments, the invention extends to a bispecific antibody, wherein the antibody of the invention or a binding fragment thereof is linked to a second antibody or binding fragment thereof having binding specificity for a second target (the target is not TNF) . This second goal preferably helps prevent TNF-mediated signaling by the TNFRl receptor. Such multivalent, bispecific or multispecific antibodies can be prepared by a variety of recombinant methods well known to those skilled in the art.

Another aspect of the invention provides a method of treating or preventing an immune-mediated condition in a canine in need thereof, the method comprising the steps of: providing a therapeutically effective amount of an antibody of the invention that specifically binds to canine TNF[alpha] or The antigen-binding fragment, and - is administered to the dog.

In certain embodiments, the antibody is a canineized antibody. In certain embodiments, a canineized antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 or at least 85% of the amino acid sequence of SEQ ID NO: a sequence of identity; and/or a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2 or having at least 85% sequence homology to the amino acid sequence of SEQ ID NO: Amino acid sequence.

In certain embodiments, a canineized antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 3; And/or a heavy chain comprising, consisting of, or consisting essentially of: selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: The amino acid sequence of the group consisting of 7 or a sequence having at least 85% amino acid identity and more preferably at least 98% identity to the amino acid sequence.

In certain embodiments, the antibody is a chimeric antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 8; and/or a heavy chain having the amine of SEQ ID NO: A base acid sequence or an amino acid sequence having at least 85% sequence homology to the amino acid sequence of SEQ ID NO: 9. In certain embodiments, a chimeric antibody comprises an amino acid sequence having a population selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22 or at least 85% with the amino acid sequence The heavy chain of the amino acid sequence of 90% or 95% sequence homology.

In certain embodiments, the immune mediated condition is a chronic inflammatory disease. The chronic inflammatory disease may be selected from the following groups (but not limited to the following): rheumatoid arthritis (RA), osteoarthritis and other polyarthritis, ankylosing spondylitis (AS), Crohn's Crohn's disease and ulcerative colitis, psoriasis and psoriatic arthritis (PsA), systemic vasculitis, atopic dermatitis, congestive heart failure (CHF), refractory uveitis, bronchial asthma and allergies Sexual condition. Inflammation-mediated conditions can also include sepsis and shock; diabetes; and neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, stroke, and amyotrophic lateral sclerosis . Monoclonal antibodies against TNF-α can also be used to prevent or treat cancer, skin tumors (such as basal cell carcinoma), colorectal cancer, and cachexia of ovarian cancer.

In certain other embodiments, the immune mediated condition can be a TNF-[alpha] related disorder or a condition in which TNF-[alpha] is a key inflammatory mediator. Such conditions include, but are not limited to, Behcet's disease, bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, systemic vasculitis, gangrene Pyoderma, pustular dermatitis, alcoholic hepatitis, cerebral malaria, hemolytic uremic syndrome, pre-eclampsia, allograft rejection, otitis media, snake bite, nodular erythema, myelodysplastic syndrome, graft resistance Host disease, dermatomyositis and polymyositis.

According to another aspect of the present invention, there is provided a method of treating an arthritis or arthritis condition in a canine individual in need thereof, the method comprising the steps of: providing a therapeutically effective amount of an anti-canine TNF antibody of the invention or antigen binding thereof Fragment, and - cast it to the dog.

In certain embodiments, the antibody is a canineized antibody. In certain embodiments, a canineized antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 or at least 85% of the amino acid sequence of SEQ ID NO: a sequence of identity; and/or a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2 or having at least 85% sequence homology to the amino acid sequence of SEQ ID NO: Amino acid sequence.

In certain embodiments, the arthritis or arthritis condition comprises a condition selected from the group consisting of: immune-mediated polyarthritis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, adolescents Idiopathic arthritis, ankylosing spondylitis, and related conditions.

Treatment of arthritis or arthritic conditions typically involves ameliorating, inhibiting, reducing or suppressing an immune response that causes an arthritic condition, is associated with an arthritic condition, or is attributable to an arthritic condition.

Another aspect of the present invention provides an increase in the performance of a canine TNF or an increase in sensitivity to TNF in a dog in need thereof, A method of treating or causing the increased condition, the method comprising the steps of: providing a therapeutically effective amount of an anti-canine TNF antibody or antigen-binding fragment thereof of the invention, and - administering it to a canine.

In certain embodiments, the above methods of the invention additionally comprise the step of co-administering at least one additional agent that enhances and/or complements the effectiveness of the anti-TNF antibody of the invention. For example, an antibody or antigen-binding fragment thereof can be co-administered with one or more other pharmaceutical compositions comprising a drug suitable for the treatment of a chronic inflammatory condition, in particular a TNF-[alpha]-related disorder. In particular, other pharmaceutical compositions may be methotrexate, soluble p55 or p75 TNF receptor or derivative thereof, chimeric or canine antibody against canine TNF, anti-canine TNF antibody fragment, at least one analgesic A compound, a compound that acts as a cytokine inhibitory anti-inflammatory drug, an NSAID, an opioid, a corticosteroid, a steroid or a nerve growth factor antagonist (such as an anti-NGF antibody).

Examples of suitable analgesics include, but are not limited to, butorphanol, buprenorphine, fentanyl, flunixin meglumine, merpidine , morphine, nalbuphine and its derivatives. Suitable NSAIDs include, but are not limited to, acetaminophen, acetaminosalicylic acid, carprofen, etodolac, ketoprofen, meloxicam ), firocoxib, robenacoxib, deracoxib, and the like.

In certain other specific embodiments, the at least one additional agent can be a therapeutically active agent, which can be one or more selected from the group consisting of an antibiotic, an antifungal, an antiprotozoal, an antiviral, and a similar therapeutic agent. Furthermore, at least one other agent may be an inhibitor of an inflamed mediator, such as a PGE receptor antagonist; an immunosuppressive agent, such as cyclosporine; or an anti-inflammatory glucocorticoid. In certain other aspects, at least one other agent can be an agent for treating a cognitive dysfunction or a cognitive disorder, such as a memory loss or related condition that can become increasingly prevalent in older dogs. Additionally, at least one other agent may be an anti-hypertensive agent or other compound for the treatment of cardiovascular dysfunction, such as for the treatment of hypertension, myocardial ischemia, congestive heart failure, and the like. Additionally, the at least one additional agent can be a diuretic, a vasodilator, a beta-adrenergic receptor antagonist, an angiotensin-II converting enzyme inhibitor, a calcium channel blocker, or an HMG-CoA reductase inhibitor.

In certain embodiments, the antibody or antigen-binding fragment is administered to the dog as part of the above method at a dose of from about 0.01 mg/kg body weight to about 10 mg/kg body weight, in particular 0.03 mg/kg body weight to Approximately 3 mg / kg body weight.

In various other aspects, the invention extends to a composition comprising an antibody of any of the above aspects of the invention, or a binding fragment thereof. In certain embodiments, the composition additionally comprises at least one pharmaceutically acceptable carrier.

Another aspect of the present invention provides a pharmaceutical composition for treating pain in a dog or causing chronic pain or a condition caused by chronic pain, comprising a pharmaceutically effective amount of the anti-canine TNF canine antibody of the present invention and at least One kind A pharmaceutically acceptable carrier, excipient or diluent.

In certain embodiments, the composition may additionally comprise methotrexate or at least one analgesic, NSAID, opioid, corticosteroid, steroid or nerve growth factor antagonist.

In various other aspects, the invention extends to isolated nucleic acids encoding an antibody or antibody binding fragment of the invention.

Accordingly, another aspect of the invention provides an isolated nucleic acid or polynucleotide encoding an antibody or antigen-binding fragment of any of the above aspects of the invention. In certain embodiments, the nucleic acid or polynucleotide encodes a light chain variable domain of an anti-canine TNF canineized antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 1, or has SEQ ID NO: The light chain of the amino acid sequence. In certain other specific embodiments, the polynucleotide encodes a heavy chain variable domain of an anti-canine TNF canine antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 2, or has a selected from SEQ ID NO: 4 The heavy chain of the amino acid sequence of the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

In certain other specific embodiments, the polynucleotide encodes a heavy chain variable domain of an anti-canine TNF chimeric antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 8, and/or has SEQ ID NO:9 The heavy chain of the amino acid sequence.

In certain embodiments, the isolated nucleic acid additionally encodes one or more regulatory sequences operably linked thereto. In another aspect, an expression vector comprising a polynucleotide encoding a heavy chain and/or a light chain variable domain or a heavy chain and/or a light chain constant domain of the invention is provided. In certain embodiments, the expression vector additionally comprises one or more regulatory sequences. In certain embodiments, the vector is a plastid or retroviral vector. Another aspect provides a host cell, It also has the above-described expression carrier of the present invention. Another aspect of the invention provides a host cell which produces an antibody of any of the above aspects of the invention.

Another aspect of the invention provides a method of producing a canineized anti-canine TNF neutralizing antibody, the method comprising the step of culturing a host cell of the above aspect of the invention such that the cell exhibits a canine anti-canine TNF neutralizing antibody.

Another aspect of the invention provides a method of producing an anti-canine TNF canineized antibody of the invention, comprising the steps of: expressing one or more light and/or heavy chains of an antibody of the invention in a suitable host cell A polynucleotide/nucleic acid or vector of the above aspect of the invention; recovering the expressed polypeptide, which can be expressed together in a host cell or separately in a different host cell; and isolating the antibody.

Another aspect of the invention provides a method of treating, ameliorating or inhibiting pain in a dog, the method comprising administering to the dog an effective amount of any of the above-described polynucleotides, antibodies or fragments of the invention.

Another aspect of the invention provides an antibody or antibody-binding fragment of any of the above aspects of the invention, or a pharmaceutical composition of the above aspect of the invention, or a nucleic acid, or any of the above aspects of the invention comprising the nucleic acid A carrier for treating or preventing pain in a dog.

In some embodiments, the pain is acute pain. In some other specific examples, the pain is chronic pain. In addition, the pain may be post-operative pain or pain caused by any procedure performed on the dog, and the procedure may include, but is not limited to, orthopedic surgery, soft tissue surgery, ovarian hysterectomy procedures, castration procedures, and the like. In certain other specific examples, the pain is chronic pain associated with cancer or a cancerous condition. In some other specific In the example, the pain is associated with rheumatoid arthritis or osteoarthritis, or by rheumatoid arthritis or osteoarthritis.

Another aspect of the invention provides an antibody or antibody-binding fragment of any of the above aspects of the invention, or a pharmaceutical composition of the above aspect of the invention, or a nucleic acid, or any of the above aspects of the invention comprising the nucleic acid A carrier for the treatment of arthritis, in particular immune-mediated polyarthritis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, adolescent idiopathic arthritis or ankylosing spondylitis.

Another aspect of the invention provides an antibody or antibody-binding fragment of any of the above aspects of the invention, or a pharmaceutical composition of the above aspect of the invention, or a nucleic acid, or any of the above aspects of the invention comprising the nucleic acid Use of a carrier for the preparation of a medicament for the treatment or prevention of chronic inflammatory diseases in dogs.

Another aspect of the invention provides an antibody or antibody-binding fragment of any of the above aspects of the invention, or a pharmaceutical composition of the above aspect of the invention, or a nucleic acid, or any of the above aspects of the invention comprising the nucleic acid Use of a carrier for the preparation of a medicament for the treatment, inhibition, amelioration or prevention of rheumatoid arthritis or osteoarthritis in dogs.

In another aspect, a cell line, or a derivative or progeny cell thereof, which produces an anti-canine TNF neutralizing monoclonal antibody or fragment thereof of the invention is provided.

Another aspect of the present invention provides a kit for treating a chronic inflammatory disease in a dog, or for treating a condition associated with pain, or for treating, ameliorating or inhibiting osteoarthritis or a rheumatoid joint Inflammation, the kit comprises any of the above-described anti-canine TNF antibodies of the invention and instructions for their use.

Another aspect of the present invention provides a diagnostic kit for detecting an anti-canine TNF monoclonal antibody in a fluid in vitro, in vitro, and in vivo for determining the concentration of the antibody. The kit can comprise any of the antibodies or binding fragments thereof of the invention. The kit may comprise instructions for use of any of the antibodies or binding fragments thereof of the invention.

After extensive experimentation, the inventors have employed D2E7 anti-human TNF antibody, an antibody that is not known to bind to canine TNF, and has surprisingly served as the basis for the production of antagonistic antibodies that specifically bind to canine TNF-[alpha]. The resulting non-immunogenic antibodies (which were not produced using standard CDR grafting techniques) were shown to exhibit high affinity for binding to canine TNF. Antibodies most specifically neutralize canine TNF biological function by inhibiting TNF binding to the cell membrane associated receptor TNFRl. In addition, the antibody has also been designed such that the framework and constant region only have residues present in the canine IgG molecule, making it less likely to produce a heterologous antibody against the antibody when administered to a dog. Therefore, the canineized antibody of the present invention is suitable for long-term administration for the treatment of chronic inflammatory diseases in dogs.

The process by which the present inventors have employed to produce the heavy and light chain variable domains of the antibodies of the present invention, such that it is known that a particular donor amino acid residue that is foreign to the canine is replaced with a canine residue at a position, based on the present invention In the analysis, the canine residue will maintain the conformation of the CDR regions and thus maintain binding specificity and affinity while reducing the presence of immunogenic epitopes, if the antibody is administered to the dog in an unaltered form, such An immunogenic epitope may result in the production of neutralizing antibodies against the antibody. In particular, the method of preparing an antibody of the invention (referred to as PETisation) comprises by immunizing a donor (eg, a human) The sequence of the framework region of the body is compared with the sequence of the antibody or antibody library derived from the canine to evaluate the suitability of the framework region of the donor antibody for administration to the dog. Although a comparison can be made between the donor sequence and a single member of the target sequence, it is clearly preferred to compare to the target sequence library as this would expand the number of natural selections at each Kabat position in the target species. This will not only increase the chance of "matching" between the donor and the target, but will also expand the choice of replacements where there is no match. Therefore, it will be possible to select a permutation having features as close as possible to the donor. When the donor sequence differs from the canine sequence at any Kabat number or corresponding position, the donor sequence is modified such that the amino acid residue is subjected to an amino acid residue known to occur naturally in the canine position Replace.

When substitution of an amino acid residue in a donor immunoglobulin framework region is desired, this substitution is typically carried out using a conservative substitution principle in which the amino acid residue is naturally present in the canine Kabat position and is supplied The substituted amino acid in the bulk sequence is replaced by an amino acid residue that is as closely related as possible in size, charge and hydrophobicity. The aim is to select substitutions that do not cause disturbance or breakage of the three-dimensional structure of the donor antibody or at least minimal disruption or fragmentation. In some cases, there will be ambiguous choices and each choice will have both advantages and disadvantages. The final decision may require three-dimensional simulations or even the performance of various alternative sequences. However, a clear preference will generally be obtained. As a result of this procedure, the change in the donor sequence is only carried out when the residue will be foreign in the target and the substituted amino acid is as closely related as possible to the amino acid to be replaced. Thus, foreign epitopes are avoided, but the overall three-dimensional structure is retained and thus also retains affinity and specificity.

Light and heavy chain constant regions are typically derived from canine (target) resistant body. If short-term inhibition of TNF is desired, the heavy chain constant domain is selected or modified such that it does not mediate downstream effector functions, ie, HCA, HCD or aglycosylated HCB or HCC. If long-term inhibition of TNF is desired, the heavy chain constant domain is selected or modified to mediate downstream effector function, i.e., HCB or HCC. Furthermore, since the substitution of framework residues is performed in a manner that does not affect the three-dimensional configuration of the CDR regions, there will be no change in binding specificity for the desired target.

There are four major IgG isotypes in humans and mice, and although the names are similar, they behave differently, including affinity for bacterial products (such as protein A and protein G), and their activation of complement-dependent cytolysis (CDC). Ability and its ability to induce killing of target cells via antibody-dependent cellular cytotoxicity (ADCC). When an antibody is designed to eliminate a target cell with its cognate antigen, such as in oncology, long-term inflammation control, or infection control, an IgG isotype with CDC and ADCC activity or an "armed" constant domain is selected. It is considered to have clinical benefit (for example, in human medical use, the human IgG1 isotype is preferably used for the above purpose). In contrast, activation of the immune system is considered undesirable in other settings, such as in relieving inflammation, pain, or autoimmunity, and thus human IgG isotypes with minimal CDC and ADCC activity are preferred (eg, In human medical applications, IgG4 isoforms are generally preferred). Four different immunoglobulin gamma (IgG) heavy chain constant domain isotypes as well as single kappa and lambda constant domain sequences have been described in the canine immune system (U.S. Patent No. 5,852,183; Tang L. et al. 2001. Veterinary Immunology and Immunopathology, 80.259- 270). Four canine heavy chain constant domains A, B, C and D have not yet been based on functional activities mediated by them Characterization. Although the IgG family is generally homologous, proteins encoding canine IgG are more related to each other than family members from other species, so it is not possible to define which of these functions (if any) can be attributed only by homology. In each of the four canine types.

The antibodies of the invention comprise a heavy chain and a light chain constant domain derived from a canine. In addition, the complementarity determining region is derived from the D2E7 antibody. The D2E7 antibody is described by Salfeld et al. (2000) and is described in US Patent 6,090,382.

The CDR regions derived from the D2E7 antibody are combined with the framework region sequences that have been determined by the inventors to retain the CDR tertiary structure, and thus retain the binding specificity, while preventing the production of heterologous antibodies against the antibody when the antibody is administered to the dog.

The light and heavy chain variable regions each contain four framework regions, designated FR1 through FR4. For each of these framework regions, the inventors have identified preferred amino acid residues (so-called preferred residues) for each particular position, and, in addition, acceptable alternatives at that position. Amino acid residue. Tables 1 to 8 illustrate four framework regions of each heavy chain and light chain. The tables provide amino acid positions relative to the particular framework region and additionally based on the Kabat numbering system used to identify the position of a particular residue along the length of the intact heavy or light chain variable domain. Amino acid residues were identified using a one-letter system.

Thus, a canineized antibody of the invention differs from, for example, a chimeric monoclonal antibody consisting of an entire variable region derived from a first species and a constant domain derived from a second species; or a canineized antibody that is CDR-grafted, wherein The complementarity determining regions (CDRs) of the variable regions of the strand and the light chain comprise an amino acid residue derived from a donor antibody and introduced into a framework region (FR) and a constant region (CR) derived from a target antibody or canine germline sequence. .

The canineized antibody preferably retains substantially the binding properties of the parent (donor) antibody from which the CDR is derived. This means that the canineized antibody will exhibit the same or substantially the same antigen binding affinity and affinity as the donor antibody from which the CDR is derived. Desirably, the affinity of the canineized antibody is not less than 10%, more preferably no less than about 30%, of the donor antibody's affinity for the target epitope, and the affinity is preferably at least the parent (donor) antibody. Affinity. Methods for analyzing antigen binding affinity are well known in the art and include half maximal binding assays, competition assays, and Scatchard analysis.

As defined above, the invention extends to a binding member or antigen-binding fragment derived from a canineized antibody of the invention. Such antigen-binding fragments refer to one or more fragments of the antibody that retain the ability to specifically bind to canine TNF. It has been shown that the antigen binding function of an antibody can be performed by a fragment of a full length antibody. In some embodiments, the binding member or antigen-binding fragment can be an isolated knot Synthesizer. A binding member or antigen-binding fragment of the invention may comprise a fragment of an antibody of the invention, such as a fragment of a fully canineized antibody molecule, such as only a heavy or light chain, or a variable domain such as a heavy chain and/or a light chain. In certain embodiments, a binding member can typically comprise, consist of, or consist essentially of an antibody VH and/or VL domain. The VH domain of the binding member is also provided as part of the present invention. There are three complementarity determining regions ("CDRs") and four related framework regions ("FR") in each VH and VL domain. The VH domain typically comprises three HCDRs (heavy chain complementarity determining regions) and the VL domain typically comprises three LCDRs (light chain complementary regions). Thus, a binding member can comprise a VH domain comprising, in turn, a VH CDR1 (or VHCDR1), a CDR2 (VHCDR2) and a CDR3 (VHCDR3) region, and a plurality of related framework regions. The binding member may additionally or alternatively comprise a VL domain comprising the VL CDR1, CDR2 and CDR3 domains and associated framework regions. The VH or VL domain typically comprises four framework regions FR1, FR2, FR3 and FR4. The term "framework region" or "framework sequence" as used herein refers to the variable region minus the remaining sequence of the CDR. Since the exact definition of a CDR sequence can be determined by a different naming system (Kabat, Chothia, etc.), the meaning of the framework sequence is subject to a correspondingly different interpretation. The six CDRs (the VLCDR1, VLCDR2 and VLCDR3 of the light chain and the VHCDR1, VHCDR2 and VHCDR3 of the heavy chain) divide the framework regions on the light and heavy chains into four sub-regions on each strand, called FR1, FR2, FR3 and FR4.

Figure 1 shows the amino acid sequence of the light chain variable domain of the anti-TNF antibody of the present invention. The CDR1, CDR2 and CDR3 regions are underlined. Therefore and as shown in Figure 1 Shown, VLCDR1 is located between the FR1 and FR2 framework regions, VLCDR2 is between the FR2 and FR3 framework regions, and VLCDR3 is located between the FR3 and FR4 framework regions. Figure 2 shows the amino acid sequence of the heavy chain variable domain of the anti-TNF antibody of the present invention. The CDR1, CDR2 and CDR3 regions are underlined. As is the case with the light chain variable region shown in Figure 1, the VHCDR1 is located between the FR1 and FR2 framework regions, the VHCDR2 is located between the FR2 and FR3 framework regions, and the VHCDR3 is located between the FR3 and FR4 framework regions.

In the table, the residues of the light chain variable domain and the heavy chain variable domain are known according to the numbering system designed by Kabat et al. (Kabat, EA, Wu, TT, Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication No. 91-3242, Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391). Kabat numbering system refers to a system for numbering amino acid residues that are relatively variable (ie, hypervariable) compared to other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof. . Thus, when referring to residues in the variable domain (approximately residues 1-104 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used. This numbering system can be used for the description in this manual. The Kabat residue name does not always correspond directly to the linear numbering of the amino acid residues of the heavy and light chain variable regions of the invention provided in the related sequences set forth herein. In particular, an actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to the framework regions or complementarity determining regions of the basic variable domain structure of the heavy or light chain, as compared to the stringent Kabat numbering ( Shortening or insertion of structural components in the CDR). By comparing the residues in the antibody sequence to the standard sequence to which Kabat numbering has been applied Yes, the correct Kabat numbering of the residues of any given antibody can be determined.

As described above, the antibody-binding fragment may be selected from the group consisting of, but not limited to, a Fab fragment, a Fab' fragment, and an scFv (single-chain variable fragment), a peptidomimetic, a bifunctional antibody, or a related multivalent derivative.

In certain embodiments, the antibody binding fragment is a Fab or F(ab')2 fragment consisting of the VL, VH, CL, and CH1 domains of the antibody. In certain embodiments, the VL domain has the amino acid sequence of SEQ ID NO: 1, and the VH domain has the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the CL and CH1 domains are based on the amino acid sequence of the CL and CH1 domains of canine immunoglobulin.

Techniques for the recombinant production of Fab, Fab' and F(ab')2 fragments are well known to those skilled in the art and include International PCT Patent Publication WO 92/22324 and Sawai et al., "Direct Production of the Fab Fragment". Derived From the Sperm Immobilizing Antibody Using Polymerase Chain Reaction and cDNA Expression Vectors", 1995, AJRI 34: 26-34. Examples of techniques that can be used to generate scFv (single-chain Fv fragments) are disclosed in Huston et al., "Protein Engineering of Single-Chain Fv Analogs and Fusion Proteins", Methods in Enzymology , Vol. 203: 46-88 (1991). Its content is incorporated by reference.

In some embodiments, the antibody fragment can be according to the method of Morimoto (Morimoto et al., "Single-step purification of F(ab') 2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl -5PW"Journal of Biochemical and Biophysical Methods 24: 107-117 (1992)) are obtained from full length antibodies by proteolytic digestion. Antibody fragments can also be produced directly by host cells (Carter et al., "High level Escherichia coli expression and production of a bivalent humanized antibody fragment" Bio/Technology 10: 163-167 (1992)).

In addition to providing a canineized monoclonal antibody that has binding specificity for canine TNF and antagonizes canine TNF function, the invention further extends to a binding member other than an antibody comprising a pair of binding domains based on SEQ ID NO The amino acid sequence of the VL (light chain variable) region as defined in 1 and the amino acid sequence of the VH (heavy chain variable) region as defined in SEQ ID NO: 2. In particular, the invention extends to a single binding domain of the VL or VH region of a canineized antibody based on an antibody of the invention.

Thus, in certain other embodiments of the invention, a binding member comprising, consisting of, or consisting essentially of a single binding domain derived from a canineized antibody of the invention is provided. In certain embodiments, a single binding domain is derived from an amino acid sequence of VH (heavy chain variable domain) as defined in SEQ ID NO:2. Such a binding domain can be used as a targeting agent for canine TNF.

In certain embodiments, other engineering techniques can be used to modify the antibodies of the invention, for example by modification comprising an Fc region that alters serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cells. toxicity. Additionally, in certain embodiments, antibodies or antibody fragments having altered glycosylation patterns can be produced. In certain embodiments, the antibodies of the invention are altered to increase or decrease the degree of glycosylation of the antibody. Polypeptide glycosylation The ground is N-connected or O-connected. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an aspartate residue. The tripeptide sequence aspartame-X-serine and aspartame-X-threonine (where X is any amino acid other than proline) is the carbohydrate moiety and the aspartate side chain The recognition sequence for enzymatic ligation. Thus, the presence of any of these tripeptide sequences in a polypeptide will result in a potential glycosylation site. O-linked glycosylation means that one of the sugars N-acetyl galactosamine, galactose or xylose is linked to a hydroxylamine acid, most often seric acid or threonine, although 5-hydroxyindole can also be used. Amino acid or 5-hydroxy lysine.

In certain other embodiments, the antibody can be PEGylated by reacting an anti-canine TNF antibody of the invention with a polyethylene glycol (PEG) derivative. In certain embodiments, the antibody removes trehalose and thus lacks trehalose residues.

In certain embodiments, changes in the biological properties of the antibody can be achieved by selective substitutions that affect (a) the structure of the polypeptide backbone in the substituted region, such as in a sheet configuration or a helical configuration; (b) target The charge or hydrophobicity of the molecule at the site; or (c) the volume of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (ALLehninger, Biochemistry, 2nd edition, 73-75, Worth Publishers, New York (1975)): (1) Non-polar: Ala (A), VaI (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) without charge, polarity: GIy (G), Ser ( S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidity: Asp (D), GIu (E); (4) Alkaline: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into the following groups based on common side chain properties: (1) hydrophobic Sex: leucine, Met, Ala, VaI, Leu, Ile; (2) neutral, hydrophilic: Cys, Ser, Thr, Asn, GIn; (3) acidity: Asp, GIu; (4) alkaline :His, Lys, Arg; (5) Residues affecting the chain position: GIy, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions require the exchange of one of these types of members with another type of member. The substituted residues can also be introduced into a conservative substitution site or introduced into a remaining (non-conserved) site.

In various other aspects, the invention extends to an immunoconjugate comprising an anti-canine TNF antibody or antigen binding portion thereof of the invention linked to a partner molecule. In certain embodiments, such antibody-complex molecular conjugates are joined by means of a chemical linker such as a peptidyl linker, a hydrazone linker or a disulfide linker. In certain embodiments, the coupling partner is an effector molecule, a label, a drug, or a carrier molecule. Suitable techniques for coupling the antibodies of the invention to peptidyl and non-peptidyl coupling partners will be well known to those skilled in the art. Examples of suitable labels include detectable labels, such as radioactive labels; or enzymatic labels, such as horseradish peroxidase; or chemical moieties, such as biotin. Alternatively, the label can be a functional marker, such as ricin; or a prodrug that is capable of converting the prodrug to the active drug at the antibody binding site.

In various other aspects, the invention extends to polynucleotides and, in particular, isolated polynucleotides encoding the canineized antibodies, antibody fragments and binding members of the invention. A "polynucleotide" as defined herein includes any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. , including (but not limited to) single and double stranded RNA, as well as single and double stranded areas The RNA of the mixture. A polynucleotide of the invention (e.g., a polynucleotide encoding a polypeptide of the invention) includes a dual gene variant thereof and/or a complement thereof, including a hybrid that hybridizes to the nucleotide sequence under moderate or high stringency conditions. Nucleotide. The invention further extends to antibody mimetics, such as domain antibodies, nanobodies, unibodies, versabody, and duocalin, which are based on the canine TNF antibodies of the invention. A variety of antibody simulation techniques are known to those skilled in the art. For example, a domain antibody (Domantis, UK) is a small functional binding unit of an antibody that corresponds to the variable region of the light or heavy chain of a human antibody. A description of the production of such domain antibodies can be found in U.S. Patent No. 6,291,158, U.S. Patent No. 6,582,915, and U.S. Patent No. 6,593,081. Nanobodies are therapeutic proteins derived from antibodies that contain the unique structural and functional properties of naturally occurring heavy chain antibodies found in camelids. A single antibody is another antibody fragment technique based on the removal of the hinge region of an IgG4 antibody. Deletion of the hinge region produces a molecule that is about one-half the size of a conventional IgG4 antibody and has a monovalent binding region. Single antibodies retain the inert nature of IgG4 antibodies and therefore do not induce an immune response.

Produce body

The antibodies and binding members of the invention can be produced, in whole or in part, by chemical synthesis. For example, antibodies and binding members of the invention can be prepared by techniques well known to those skilled in the art, such as standard liquid peptide synthesis or solid phase peptide synthesis methods. Alternatively, antibodies and binding members can be prepared in solution using liquid phase peptide synthesis techniques, or additionally by solid phase, liquid phase, and solution chemistry. The combination is prepared.

The invention further extends to the production of an antibody or binding member of the invention by expressing a nucleic acid encoding at least one amino acid comprising an antibody of the invention in a suitable expression system such that the desired peptide or polypeptide can be encoded. For example, a nucleic acid encoding an amino acid light chain and a second nucleic acid encoding an amino acid heavy chain can be represented to provide an antibody of the invention.

Thus, in certain other aspects of the invention, nucleic acids encoding amino acid sequences that form an antibody or binding member of the invention are provided.

A nucleic acid encoding an amino acid sequence that forms an antibody or binding member of the invention will typically be provided in isolated or purified form, or in a form substantially free of a substance with which it may be naturally associated, with the exception of one or more regulatory sequences. provide. Nucleic acids that exhibit antibodies or binding members of the invention may be fully or partially synthetic and may include, but are not limited to, DNA, cDNA, and RNA.

Nucleic acid sequences encoding the antibodies or binding members of the invention can be readily prepared by the skilled artisan using techniques well known to those skilled in the art, such as Sambrook et al. "Molecular Cloning", A laboratory manual, cold Spring Harbor Laboratory Press. , Techniques described in Volumes 1 to 3, 2001 (ISBN-0879695773) and Ausubel et al., Short Protocols in Molecular Biology. John Wiley and Sons, 4th Edition, 1999 (ISBN-0471250929). Such techniques include (i) polymerase chain reaction (PCR) to amplify a sample of nucleic acid, (ii) chemical synthesis, or (iii) preparation of a cDNA sequence. DNA encoding an antibody or binding member of the invention can be produced and used in any suitable manner known to those skilled in the art, including performing DNA coding to identify portions to be expressed. The restriction enzyme recognition site is suitable for either side and is excised from the DNA. The excised portion can then be operatively coupled to a suitable initiator and rendered in a suitable performance system, such as a commercially available performance system. Alternatively, the relevant portion of the DNA can be amplified by using a suitable PCR primer. Modification of the DNA sequence can be induced by using site-directed mutagenesis.

A nucleic acid sequence encoding an antibody or binding member of the invention may be provided as a construct in a form comprising at least one of a plastid, vector, transcription or expression cassette of a nucleic acid as described above. The construct can be included in a recombinant host cell comprising one or more of the above constructs. Performance is preferably achieved by culturing a recombinant host cell containing a suitable nucleic acid sequence under appropriate conditions. Following performance, the antibody or antibody fragment can be isolated and/or purified using any suitable technique, and then used as appropriate.

Systems for the selection and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, insects, and baculovirus systems. Mammalian cell lines useful in the art for expressing heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, and NSO mouse myeloma cells. A preferred bacterial host is Escherichia coli. The expression of antibodies and antibody fragments in prokaryotic cells, such as E. coli, is well established in the art. Performance in eukaryotic cells in culture can also be utilized by those skilled in the art as a means of generating binding members.

The general techniques for producing antibodies are well known to those skilled in the art, and such methods are discussed, for example, in Kohler and Milstein (1975) Nature 256:495-497; U.S. Patent No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor. Techniques for the preparation of recombinant antibody molecules are described in the above references and in, for example, European Patent No. 0,368,684.

In certain embodiments of the invention, recombinant nucleic acids comprising an insert encoding a heavy chain variable domain and/or a light chain variable domain of an antibody or binding member are employed. By definition, the nucleic acids comprise a single-stranded nucleic acid, a double-stranded nucleic acid consisting of such an encoding nucleic acid and its complementary nucleic acid, or such complementary (single-stranded) nucleic acid itself.

Furthermore, the nucleic acid encoding the heavy chain variable domain and/or the light chain variable domain of the antibody may be an enzymatically or chemically synthesized nucleic acid or a mutant thereof, which encodes a naturally occurring heavy chain variable domain and/or Or the authentic sequence of the light chain variable domain.

The antibody of the present invention can be produced not only directly by recombinant means, but also as a fusion polypeptide which is preferably a heterologous polypeptide of a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably a heterologous signal sequence that can be recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is substituted with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, lpp or a heat stable enterotoxin II leader.

The term "isolated" when used in reference to a canineized antibody of the invention, or a binding member derived therefrom, or a polynucleotide encoding the same, refers to the following states: such antibodies, binding members or nucleic acids ( Polynucleotide) is provided in isolated and/or purified form, ie it has been isolated from its natural environment Is isolated, isolated or purified; and provided in substantially pure or homogeneous form; or in the case of a nucleic acid, in the absence or substantial absence of a nucleic acid or gene form other than a sequence encoding a polypeptide having the desired function provide. Thus, the isolated antibodies, binding members, and isolated nucleic acids will be free or substantially free of materials with which they are naturally associated, such as other polypeptides or nucleic acids that are present together in the environment: their natural environment; or their preparation environment (e.g., cell culture), wherein the preparation is carried out by recombinant DNA techniques carried out in vitro or in vivo.

Antibodies, binding members, and nucleic acids can be formulated with diluents or adjuvants, and are still considered to be provided in isolated form for practical purposes. For example, if antibodies and binding members are used to coat microtiter dishes for immunoassays, such antibodies and binding members can be mixed with gelatin or other carriers; or when used in diagnosis or therapy, The antibodies and binding members will be combined with a pharmaceutically acceptable carrier or diluent. The antibody or binding member can be glycosylated, either naturally or by a system of heterologous eukaryotic cells (e.g., CHO or NSO cells), or it can be ungyolated (e.g., if produced by expression in prokaryotic cells).

Heteroconjugates comprising anti-canine TNF canine antibody molecules also form part of the invention. For example, such preparations may be heavy chains having a full length heavy chain and lacking a C-terminal lysine, having various degrees of glycosylation, and/or having a derivatized amino acid (such as N-terminal glutamic acid cyclization to form a coke A mixture of antibodies to glutamic acid residues).

Pharmaceutical composition

The pharmaceutical composition of the present invention is typically formulated into a liquid formulation, lyophilized Formulations, lyophilized formulations or aerosol formulations that are reconstituted in liquid form. In certain embodiments, the concentration of the antibody in the formulation is as follows: from about 0.5 mg/ml to about 250 mg/ml, from about 0.5 mg/ml to about 45 mg/ml, from about 0.5 mg/ml to about 100 mg/ M, from about 100 mg/ml to about 200 mg/ml or from about 50 mg/ml to about 250 mg/ml.

In certain embodiments, the formulation additionally comprises a buffer. The pH of the formulation is typically from about pH 5.5 to about pH 6.5. In certain embodiments, the buffer can comprise from about 4 mM to about 60 mM histidine buffer, from about 5 mM to about 25 mM succinate buffer, or from about 5 mM to 25 mM acetate buffer. In certain embodiments, the buffer comprises sodium chloride at a concentration of from about 10 mM to 300 mM, typically at a concentration of about 125 mM, and sodium citrate at a concentration of from about 5 mM to 50 mM, typically 25 mM. . In certain embodiments, the formulation may additionally comprise a surfactant having a concentration of only greater than 0% to about 0.2%. In certain embodiments, the surfactant is selected from the group consisting of, but not limited to, polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65 , Polysorbate-80, Polysorbate-85, and combinations thereof. In a preferred embodiment, the surfactant is polysorbate-20 and may additionally comprise sodium chloride at a concentration of about 125 mM and sodium citrate at a concentration of about 25 mM.

Dosing

The antibody or binding member of the present invention may be administered alone, but preferably will be administered in the form of a pharmaceutical composition which will generally comprise a suitable pharmaceutically acceptable excipient selected according to the intended route of administration, diluted. Agent or carrier. Examples of suitable pharmaceutical carriers include: water, glycerin, ethanol, and the like. Things.

The monoclonal antibodies or binding members of the invention can be administered to a canine patient in need of treatment via any suitable route. The composition is typically administered parenterally by injection or infusion. Examples of preferred parenteral routes of administration include, but are not limited to, intravenous, intracardiac, intraarterial, intraperitoneal, intraarticular, intramuscular, intraluminal, subcutaneous, transmucosal or transdermal. The route of administration may additionally include topical and enteral administration, such as transmucosal (including transpulmonary), oral, nasal or rectal.

In specific embodiments where the composition is delivered as an injectable composition, such as intravenous, intradermal or subcutaneous administration, the active ingredient may be pyrogen-free and have a suitable pH, isotonicity and stability. Acceptable aqueous form. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as sodium chloride injection, Ringer' injection or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and/or other additives may be included as needed.

Compositions can also be administered via microspheres, liposomes, other microparticle delivery systems, or sustained release formulations placed in certain tissues, including blood.

Examples of the techniques and protocols mentioned above, as well as other techniques and protocols that can be used in accordance with the present invention, can be found in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro, AR, Lippincott Williams &Wilkins; 20th Edition ISBN 0-912734-04 -3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, HC et al., 7th Edition, ISBN 0-683305-72-7, the entire disclosure of which is incorporated herein by reference.

The antibodies and compositions of the present invention are typically administered to a subject in a "therapeutically effective amount" which is an amount sufficient to provide an benefit to the individual to whom the composition is administered. The actual dose administered and the rate of administration will be determined as follows and can be determined by reference to the nature and severity of the condition being treated, as well as factors such as the age, sex and weight of the individual being treated, and the route of administration. . In addition, consideration should be given to the nature of the composition, such as its binding activity and in vivo plasma life, the concentration of antibody or binding member in the formulation, as well as the route, site and rate of delivery.

Dosing regimens can include a single administration of an antibody or composition of the invention, or multiple doses of an antibody or composition. The antibody or antibody-containing composition may be administered separately or separately from other therapeutic agents and pharmaceuticals for treating the condition in which the antibody or binding member of the present invention is administered for treatment.

Examples of dosing regimens that can be administered to an individual can be selected from the group consisting of, but not limited to, the following: 1 microgram/kg/day to 20 mg/kg/day, 1 microgram/kg/day to 10 mg/kg/day. , 10 μg / kg / day to 1 mg / kg / day. In certain embodiments, the dosage will be a dose that achieves a plasma concentration of antibody from 1 μg/ml to 100 μg/ml. However, the actual dosage of the composition administered, as well as the rate and timing of administration, will depend on the nature and severity of the condition being treated. The treatment site (eg, dose determination, etc.) is ultimately within the responsibilities of the veterinary practitioner and other veterinarians and is determined by the veterinary practitioner and other veterinarians, and typically considers the condition being treated, the condition of the individual patient, and the site of delivery. , methods of administration, and other factors known to practitioners.

definition

Unless otherwise defined, all technical and scientific terms used herein have the meanings The meaning and scope of the terms should be clear, however, if there is any ambiguity, the definitions provided herein take precedence over any dictionary or foreign definition.

Throughout the specification, the terms "comprise" or "include" are to be understood to mean the inclusion of the whole or whole group, but does not exclude any other whole or whole group. .

Terms such as "a" and "the", as used herein, include the singular and plural referents. Thus, for example, reference to "an active agent" or "a pharmacologically active agent" includes a single active agent and two or more different active agents in a combined form, Reference to a "carrier" includes a mixture of two or two of a carrier and a single carrier, and the like. In addition, unless otherwise required, the singular terms shall include the plural and the plural terms shall include the singular.

The term TNF neutralizing antibody or like terms as defined herein describes an antibody that is capable of neutralizing the biological activity and signaling of TNF. Neutralizing antibodies, also known as antagonistic or blocking antibodies, specifically and preferably selectively bind to TNF and inhibit one or more biological activities of TNF. For example, neutralizing antibodies can inhibit TNF binding to its target ligand, such as the cell membrane-bound TNF receptor 1 (TNFRl) receptor (CD120a).

The term "biological activity" as used herein refers to any one or more of the intrinsic biological properties of a molecule (whether naturally occurring in In vivo, or provided or implemented by recombinant means). Biological properties include, but are not limited to, receptor binding and/or activation, induction of cell signaling or cell proliferation, inhibition of cell growth, induction of cytokine production, induction of apoptosis, and enzymatic activity.

The term "complementarity determining region (CDR)" as used herein refers to an amino acid sequence that collectively defines the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site, such as Kabat et al. (Kabat). , EA, Wu, TT, Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th Edition. NIH Publication No. 91-3242). The term framework region (FR) as used herein refers to an amino acid sequence inserted between CDRs. These antibody moieties are used to keep the CDRs in the proper orientation (allowing the CDRs to bind to the antigen).

The term "constant region (CR)" as used herein refers to a portion of an antibody molecule that confers an effector function. In the present invention, the constant region typically means a canine constant region, i.e., the constant region of the canineized antibody of the present invention is derived from canine immunoglobulin. The heavy chain constant region can be selected from any of the four isotypes A, B, C or D.

The term "chimeric antibody" as used herein refers to an antibody comprising sequences derived from two different antibodies derived from different species. A chimeric antibody most typically comprises a variable domain derived from a specified donor and specifically binding to a target epitope, and a constant domain derived from an antibody obtained from a target species to which the antibody is to be administered.

The term "immunogenicity" as used herein. Refers to a measure of the ability of a targeted protein or therapeutic moiety to elicit an immune response (body fluid or cell) when administered to a recipient. The present invention relates to the immunogenicity of the canineized antibodies of the invention. Preferably, the antibodies of the invention are not immunogenic, i.e., will not produce a heterologous antibody against the antibodies when administered to a dog.

The term "identity" or "sequence identity" as used herein means the amino acid residue between aligned sequences at the position of any particular amino acid residue in the aligned sequence. Consistent. The term "similarity" or "sequence similarity" as used herein indicates that the amino acid residues between the sequences belong to a similar type at any particular position in the aligned sequence. For example, leucine can replace an isoleucine or a valine residue. This substitution can be referred to as a conservative substitution. When the amino acid sequence of the present invention is modified by conservative substitution of any of the amino acid residues contained therein, such changes preferably have binding specificity or function to the resulting antibody as compared to the unmodified antibody. Activity has no effect.

Sequence identity relative to a (native) polypeptide of the invention and a functional derivative thereof refers to the alignment of the sequence and, if necessary, the introduction of a gap to achieve a maximum percent homology, without considering any conservative substitution as sequence identity In the case of a portion, the percentage of amino acid residues in the candidate sequence that are identical to the residues of the corresponding native polypeptide. N-terminal or C-terminal elongation or insertion should not be considered to reduce sequence identity or homology. Methods and computer programs for performing alignment of two or more amino acid sequences and determining their sequence identity or homology are well known to those skilled in the art. For example, the percentage of identity or similarity of two amino acid sequences can be readily calculated using an algorithm such as BLAST (Altschul et al. 1990), FASTA. (Pearson and Lipman 1988) or Smith-Waterman algorithm (Smith and Waterman 1981).

As used herein, reference to an amino acid residue having "highest homology" with respect to a second amino acid residue refers to an amine having the most common characteristics or characteristics with the second amino acid residue. Base acid residue. In determining whether the amino acid residue has the highest homology to the second amino acid residue, the following factors such as, but not limited to, charge, polarity, hydrophobicity, side arm mass, and side arm size can typically be evaluated. .

The term "corresponding position" as used herein in relation to an amino acid residue at a position corresponding to a specified amino acid residue in the first sequence in the second sequence is intended to mean when When the sequence is such that the maximum sequence identity is reached between the two sequences, the position in the second sequence is at the same position as the position in the first sequence. The amino acid residues at the corresponding positions have the same Kabat number.

The term "consist essentially of" as used herein means that the polypeptide may have other features or elements other than the features or elements, with the proviso that the other features or elements are not essential in nature. Affects the ability of an antibody or antibody fragment to have binding specificity in canine TNF. That is, the antibody or antibody fragment comprising the polypeptide may have additional features or elements that do not interfere with the ability of the antibody or antibody fragment to bind to canine TNF and antagonize canine TNF functional activity. Such modifications can be introduced into the amino acid sequence to reduce the immunogenicity of the antibody. For example, a polypeptide consisting essentially of a specified sequence may contain 1, 2, 3, 4, 5 or more amino acid residues or substitutions at either or both ends of the sequence, the Amino group The acid does not interfere with, inhibit, block or disrupt the binding of the antibody or fragment to canine TNF and chelate its biological function. Similarly, a polypeptide molecule that contributes to the canine TNF antagonist antibody of the present invention can be chemically modified by one or more functional groups, with the proviso that the functional groups do not interfere with the binding of the antibody or antibody fragment to canine TNF and antagonize its function. Ability.

The term "effective amount" or "therapeutically effective amount" as used herein means an agent of the invention, a binding compound, a small molecule, a fusion protein, or the like, which inhibits the binding of canine TNF to a TNFR1 receptor. The amount of peptidomimetic.

The terms "polypeptide", "peptide" or "protein" are used interchangeably herein to refer to an amine that is linked to each other by a peptide bond between an a-amino group and a carboxyl group of an adjacent residue. A linear series of base acid residues. Amino acid residues are typically in the natural "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue as long as the polypeptide retains the desired functional properties.

An "antibody" as defined herein encompasses an antigen binding protein that specifically binds to a related target antigen (in this case, canine tumor necrosis factor), which has one or more of which can be prepared recombinantly or by immunoglobulin A protein gene or a polypeptide genetically encoded by a fragment of an immunoglobulin gene. The term "antibody" encompasses both monoclonal antibodies and chimeric antibodies (in detail canineized antibodies) and additionally encompasses multiple antibodies or antibodies of any kind or subtype. "Antibodies" are further extended to hybrid antibodies, bispecific antibodies, heterologous antibodies, and functional fragments that retain antigen binding.

The term "specifically binds to" means resistance The body binds to a specific protein or target present in a heterogeneous population of proteins. Thus, when present under specific immunoassay conditions, the antibody binds to a particular protein (in this case, canine TNF) and does not bind in large amounts to other proteins present in the sample.

A "canine" as defined herein may also be referred to as a dog. Dogs can be classified into subspecies with three French name dogs ( Canis lupus familiaris/Canis familiaris domesticus ) or wild dogs ( Canis lupus dingo ). Dogs include any type of dog and include wild and pet breeds, the latter also known as companion animals.

The term "xenoantibody" as defined herein refers to an antibody produced by a host that is a foreign epitope to the host.

The invention will now be described with reference to the following examples, which are provided for the purpose of illustration and are not intended to limit the invention. The methods and techniques of the present invention are generally carried out in accordance with the teachings of the <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

Example

Example 1 - Expression of a DNA encoding a canine PET (PETised) anti-canine TNF MAb

The sequences of SEQ ID NO: 1 to SEQ ID NO: 9 were designed and constructed using oligonucleotide synthesis, and subcloned into pcDNA3.1+ expression vector, and transfected into CHO cells in various combinations. .

The cDNA encoding the following was subcloned into pcDNA3.1+ (Invitrogen/Life technologies): Canineized anti-TNF monoclonal antibody having the amino acid sequence of SEQ ID NO: 3 (light chain) and SEQ ID NO: 5 (heavy chain); and chimeric anti-TNF monoclonal An antibody having a light chain having the amino acid of SEQ ID NO: 8 and a heavy chain of SEQ ID NO: 9. Chimeric CHO cells with the canineized heavy and light chain sequences of SEQ ID NO: 3 and SEQ ID NO: 5 (ca-HCB+ca-kLC) or SEQ ID NO: 8 and SEQ ID NO: A combination of heavy and light chains (ch-HCB+ch-kLC) was co-transfected. The resulting supernatant was purified on Protein A, analyzed by SDS-PAGE (Fig. 10A), and coated with canine TNFa at a specified concentration (μg/ml) of the antibody by ELISA (5 μg/ml; R&D) The combination of systems was detected using an anti-canine polyclonal antibody-horseradish peroxidase conjugate (Sigma A9042) (Fig. 10B). The negative control group was multi-strain detection only on the coated antigen.

Example 2 - Inhibition of canine TNF activity

The ability of purified antibodies to inhibit canine TNF activity was tested using pTRH1 transfected 293-HEK cells to generate a TNF-sensitive NF-kB-EGFP reporter cell line that responds to human TNF by fluorescence (Vince Et al., Cell 131, 682, 2007). It was first confirmed that canine TNF activated GFP expression in these cells, and then the ability of the canine antibody shown in Fig. 11 to inhibit 1 ng/mL canine TNF was tested.

As shown in Figure 11, canineized antibodies produced by co-transfection of SEQ ID NO: 3 and SEQ ID NO: 5 (ca-HCB + ca-kLC) and by transfection of SEQ ID NO: 8 and SEQ The chimeric antibody produced by ID NO: 9 (ch-HCB+ch-kLC) is a potent inhibitor of canine TNF in this assay.

These results collectively show the canineized antibody of the present invention and the human-canine chimera The body binds to canine TNF and is equivalent in both ELISA and inhibition assays, confirming that the canineization process has produced a fully active canine form of the original D2E7 antibody.

Figure 12 (a) illustrates the comparison of canineized and chimeric D2E7 monoclonal antibody (MAb) with another canineized antibody based on anti-human TNF MAb pure line 148. Canineized anti-huTNF MAb 148 (SEQ ID NO: 18 (ca148-HCB) and SEQ ID NO: 19 (ca148-kLC)) were expressed in CHO cells and purified using Protein A chromatography (panel A, left lane). Binding of MAb to human TNF (Panel B) and canine TNF (Panel C) compared to D2E7-based canine (Ca) and chimeric (Ch) MAbs from Figures 10 and 11 (background negative control combination Shown by the arrow). As can be seen from panel B and panel C, canineized MAb 148 binds to human TNF (panel B) but does not bind to canine TNF (panel C). Thus, the D2E7-based canineized and chimeric MAb and the subject matter of the present invention surprisingly showed strong binding to canine TNF comparable to human TNF binding, whereas canineized MAb 148 showed binding only to human TNF. Therefore, D2E7-based canineized MAb is surprisingly suitable for the treatment of canine TNF-mediated canine disease. Figure 12 (b) and Figure 12 (c) illustrate the amino acid sequence of the heavy chain of canineized MAb 148 (SEQ ID NO: 18) and the amino acid sequence of the light chain of canineized MAb 148, respectively (SEQ ID NO: 19).

Figure 13 illustrates that canineized D2E7 monoclonal antibody mediates the activity of complement-killing TNF-expressing cells. RAW cells were treated with 10 ng/ml LPS (columns 1-3) or untreated (columns 4-6) to induce membrane-bound and secreted TNF expression. The cells were then treated with serum in the presence or absence of 10 μg/ml canineized anti-TNF antibody (ca-HCB+ca-kLC). Columns 1 and 4, serum and antibody; columns 2 and 5, serum only; columns 3 and 6, serum-free or antibody-free. Cell killing was assessed by counting dead cells stained with Trypan Blue. Specific cell killing was observed only in the case of a combination of canineized antibody, serum and LPS treatment (cylinder 1). As can be seen in Figure 13, subsequent treatment with a combination of canineized antibody and serum induced specific cell death (measured by trypan blue staining), whereas antibodies or serum alone did not. Injection of these antibodies into dogs will reduce the number of canine TNF-expressing cells in vivo and produce a long-term anti-inflammatory effect.

All documents mentioned in this specification are hereby incorporated by reference. Various modifications and alterations to the specific embodiments of the invention described herein will become apparent to those skilled in the art. Although the invention has been described in connection with the specific preferred embodiments, it should be understood that the claimed invention In fact, the present invention is intended to cover various modifications of the modes of the invention described herein.

Figure 1 is a SEQ ID NO: 1 showing the amino acid sequence of the light chain variable domain of the canineized antibody of the present invention.

Figure 2 is a SEQ ID NO: 2 showing the amino acid sequence of the heavy chain variable domain of the canineized antibody of the present invention.

Figure 3 is a SEQ ID NO: 3 showing the amino acid sequence of the light chain of the canineized antibody of the present invention.

Figure 4 shows the amino acid sequence (SEQ ID NO: 4) of canineized anti-TNF heavy chain variable domain canine IgG-A heavy chain (caN-HCA). Variable domain residues are shown in bold.

Figure 5 shows the amino acid sequence of the canineized anti-TNF heavy chain variable domain canine IgG-B heavy chain (caN-HCB) (SEQ ID NO: 5). Variable domain residues are shown in bold.

Figure 6 shows the amino acid sequence (SEQ ID NO: 6) of canineized anti-TNF heavy chain variable domain canine IgG-C heavy chain (caN-HCC). Variable domain residues are shown in bold.

Figure 7 shows the amino acid sequence of the canineized anti-TNF heavy chain variable domain canine IgG-D heavy chain (caN-HCD) (SEQ ID NO: 7). Variable domain residues are shown in bold.

Figure 8 shows the amino acid sequence of the canine kappa light chain of chimeric anti-TNF antibody (SEQ ID NO: 8). Variable domain residues are shown in bold.

Figure 9 shows the amino acid sequence of the heavy chain variable domain canine IgG-B heavy chain (caN-HCB) of the chimeric antibody (SEQ ID NO: 9). Variable domain residues are shown in bold.

Figure 10 (A) shows canineization (SEQ ID NO: 3 and SEQ ID NO: 5) and chimerism (SEQ ID NO: 8 and SEQ ID NO: 9), which were purified using Protein A and analyzed by SDS-PAGE. The results of the antibodies; and Figure 10 (B) shows the results of the ELISA showing that the expressed recombinant protein binds to canine TNF-[alpha]. Show results using multiple antibody dilutions from 5 μg/ml to 0.05 μg/ml.

Figure 11 shows inhibition of canine TNF biological activity using 293-HEK cells transfected with the NF-kB-EGFP reporter construct pTRH1. These cells respond to TNF by fluorescein. Canineization (SEQ ID NO: 3 and SEQ ID NO: 5) (Fig. 11A) and chimerism (SEQ ID NO: 8 and SEQ ID NO: 9) (Fig. 11B) MAbs all equally inhibited TNF-induced fluorescence (quantified in Figure 11C).

Figure 12 (a) shows a comparison with another canineized MAb based on anti-TNF MAb pure line 148 (Figure A, left lane) expressed in CHO cells and purified using Protein A chromatography. The binding of MAb to human TNF (Panel B) and canine TNF (Panel C) was compared to D2E7-based canine (Ca) and chimeric (Ch) MAb from Figure 10 (background negative control combined by arrow) ). As can be seen, D2E7-based canineized and chimeric MAbs and the subject matter of the present invention surprisingly showed strong binding to canine TNF comparable to human TNF binding, whereas canineized MAb 148 showed binding only to human TNF.

Figure 12 (b) is SEQ ID NO: 18 showing the amino acid sequence of the heavy chain of canineized MAb 148.

Figure 12 (c) is SEQ ID NO: 19 showing the amino acid sequence of the light chain of canineized MAb 148.

Figure 13 shows that canineized anti-TNF monoclonal antibodies constructed by co-expression of SEQ ID NO: 3 and SEQ ID NO: 5 are capable of inducing complement-mediated TNF-expressing cytolysis.

<110> NVIP Pty Ltd Gillen David (Gearing, David)

<120> Tumor necrosis factor antibody and method of using same

<130> P120969.WO.01

<150> US 61/529020

<151> 2011-08-30

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<170> PatentIn version 3.5

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Claims (56)

A caninised antibody or antigen-binding fragment thereof that specifically binds to canine tumor necrosis factor. The canineized antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-system is prepared by a method comprising: providing a donor antibody from a species other than a dog, wherein the donor antibody is resistant to tumor necrosis The factor has binding specificity; each amino acid residue of the amino acid sequence of the framework region of the donor antibody is at a corresponding position in the amino acid sequence of the framework region of one or more canine antibodies Amino acid residues are compared to identify one or more amines at corresponding positions in the amino acid sequence of the framework region of the one or more canine antibodies in the amino acid sequence of the framework region of the donor antibody One or more amino acid residues different in base acid residues; and substituting one of the donor antibodies with one or more amino acid residues present at corresponding positions in the one or more canine antibodies Or a plurality of identified amino acid residues. The antibody or antigen-binding fragment thereof of claim 2, wherein the step of substituting the one or more identified amino acid residues comprises using one or more amino acid residues present at the corresponding position Substituting the one or more identified amino acid residues, the one or more amino acid residues have the highest homology to the one or more substituted amino acid residues. An antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the method further comprises replacing the heavy chain of the donor antibody with a constant domain derived from a heavy chain and/or a light chain of a canine antibody and/or Step of the constant domain of the light chain Step. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 1 An amino acid sequence or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 1; and/or a heavy chain variable region comprising SEQ ID NO: 2 The amino acid sequence or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 2. The antibody or antigen-binding fragment of claim 5, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 3 or at least 85 with the amino acid sequence of SEQ ID NO: a % identical amino acid sequence; and/or a heavy chain comprising an amine group selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. An acid sequence or an amino acid sequence having at least 85% sequence identity to the amino acid sequence. The antibody or antigen-binding fragment of any one of clauses 1 to 5, wherein the antibody comprises a heavy chain comprising SEQ ID NO: 4, aglycosylated SEQ ID NO: 4, aglycosylated SEQ ID NO: 5, aglycosylated SEQ ID NO: 6, aglycosylated SEQ ID NO: 7 and SEQ ID NO: 7 amino acid sequence, or The amino acid sequence has an amino acid sequence of at least 85% sequence identity. The antibody or antigen-binding fragment of any one of clauses 1 to 5, wherein the antibody comprises a heavy chain comprising a population selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: Amino acid sequence or with the amine group The acid sequence has an amino acid sequence with at least 85% sequence identity. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment comprises a light chain variable region comprising at least one of the following: FR1 A framework region consisting of or comprising the amino acid sequence of SEQ ID NO: 10, the FR2 framework region, consisting of the amino acid sequence of SEQ ID NO: 11 or comprising SEQ ID NO: Amino acid sequence of 11 , FR3 framework region, consisting of or comprising the amino acid sequence of SEQ ID NO: 12, and the FR4 framework region, the amino group of SEQ ID NO: The acid sequence consists of or comprises the amino acid sequence of SEQ ID NO: 13; and/or the heavy chain variable region comprising at least one of the following: the FR1 framework region, the amino group of SEQ ID NO: The acid sequence consists of or comprises the amino acid sequence of SEQ ID NO: 14, the FR2 framework region, consists of the amino acid sequence of SEQ ID NO: 15 or comprises the amino acid sequence of SEQ ID NO: 15, the FR3 framework region, The amino acid sequence of SEQ ID NO: 16 consists of or comprises the amino acid sequence of SEQ ID NO: 16, and the FR4 framework region, SEQ ID NO: 17 Or comprises amino acid sequence of SEQ ID NO: 17 of the amino acid sequence. An antibody or antigen-binding fragment thereof according to claim 9, which comprises a light chain variable domain having the FR1 region of SEQ ID NO: 10, which has been selected from one or more selected from the group consisting of Repair with amino acid Decoration: amino acid residue T (T5) at position 5 is replaced by amino acid residue M or I, S7 is T, A9 is L or P, S12 is A, L13 is V, and S14 is T or R. Q15 is P or R, E16 is D, K18 is E, A, P, T or L, V19 is A, T20 is S, T22 is S or Y, and C23 is Y. An antibody or antigen-binding fragment thereof according to claim 9 or 10, which comprises a light chain variable domain having the FR2 region of SEQ ID NO: 11, which has been selected from one or more selected from the group consisting of The group of amino acids is substituted and modified: Y2 is F, I or L, Q3 is R, L or I, Q4 is H, K5 is R, P6 is S or A, G7 is D, and A9 is S, T Or P, K11 is Q, E or R, L12 is R, P, G, A or S, I14 is L, and Y15 is F, N, S, E or V. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 11, which comprises a light chain variable domain having the FR3 region of SEQ ID NO: 12, which has been utilized by one or more One is modified by an amino acid selected from the group consisting of G1 as A, V2 as A, P3 as S, S4 as D, F6 as L or V, S7 as I, G8 as A, and T13 as A, D14. E, T16 is S or R, L17 is F, T18 is R or K, S21 is R, G or T, L22 is V, P24 is A, E25 is D, G, I or N, and V27 is A, T , G or S, A28 is G, and V29 is I or L. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 12, which comprises a light chain variable domain having the FR4 region of SEQ ID NO: 13, which has been made by one or more One is modified by substitution with an amino acid consisting of: G2 is S, Q3 is A, P or T, G4 is E, T5 is P, K6 is Q or S, V7 is L or W, and E8 is D. Or R, and I9 Is L. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 13, which comprises a heavy chain variable domain having the FR1 region of SEQ ID NO: 14, which has been made by one or more The modification is carried out by an amino acid selected from the group consisting of E1 being D or G, V2 being G, L, E, I or M, and Q3 being H, R, A, V, E, K, L, P Or S, L4 is V or P, V5 is A, L, E or M, E6 is Q or A, S7 is F, L or T, G9 is E, G10 is D, A, N, E or T, L11 For Q, R, V or W, V12 is A, I or M, Q13 is K, R or N, P14 is F or T, G15 is A, E or T, G16 is E or A, and S17 is T or P. L18 is R, R19 is K, T, G or V, L20 is I or V, S21 is Y, A23 is V, L, I or E, A24 is T, V, G, I or S, and S25 is P. , G or T, G26 is D, R or T, and F27 is L, I, S, D, T or V. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 14, which comprises a heavy chain variable domain having the FR2 region of SEQ ID NO: 15, which has been made by one or more The modification is carried out by an amino acid selected from the group consisting of W1 being C, V2 being I, A, F or L, Q4 being L or H, and A5 being S, T, G, P, V or D, P6 L, G7 is E, R or L, K8 is R, E, G, A, M or Q, G9 is E, R, D, T or V, L10 is T, P, F or M, and E11 is Q , H, D, L, P or R, W12 is L, C, S, Y, F or M, V13 is L, I or F, and S14 is A, T, G or L. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 15, which comprises the weight of the FR3 region having SEQ ID NO: a chain variable domain, which has been modified by substitution of one or more amino acids selected from the group consisting of R1 being Q, F2 being V or L, T3 being A, I or S, and I4 being V , L, M or T, S5 is A, F or T, R6 is K, D7 is E or N, N8 is D, T, S, I or G, and A9 is G, V, S, D, P or T , K10 is R, E, N, Q, G or M, N11 is D, S, K, H or R, S12 is T, M, I or A, L13 is V, M, A or I, Y14 is F , H, S or T, L15 is I, Q16 is H, E, D, R or A, M17 is L, N18 is D, S, T, H, K, P or R, and S19 is G, D, R , N or T, L20 is V, R21 is T, G, K, S or I, A22 is V, D, T, S, G or P, E23 is D, A or V, and T25 is A, S or M , A26 is G or V, V27 is M, I, L, F, T, K or Q, Y28 is H, Y29 is F or H, and A31 is V, T, G, M, R, S, C or L And K32 is R, S, N, G, A, T, P, D, Q, V, E, I or M. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 16, which comprises a heavy chain variable domain having the FR4 region of SEQ ID NO: 17, which has been made by one or more The modification is carried out by an amino acid selected from the group consisting of W1 being L, G2 being A or S, Q3 being P, H, R or D, T5 being A, S, I or N, and L6 being S, Q , P or R, V7 is L, I or P, T8 is F, I, A, S, L, P or Y, V9 is A, S10 is A, C, P or T, and S11 is L, A or P. A chimeric antibody or antigen-binding fragment thereof, which specifically binds to canine tumor necrosis factor, wherein the antibody or antigen-binding fragment comprises a light chain comprising the amino acid sequence of SEQ ID NO: 8 or with SEQ ID NO The amino acid sequence of 8 has an amino acid sequence of at least 85% identity. The antibody or antigen-binding fragment of claim 18, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 or the amino acid of SEQ ID NO: The sequence has an amino acid sequence of at least 85% identity. The antibody or antigen-binding fragment of claim 18, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising a gene selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: The amino acid sequence of the group or the amino acid sequence having at least 85% identity to the amino acid sequence. The antibody or antigen-binding fragment according to any one of claims 1 to 20, wherein the antibody or antigen-binding fragment is bound by a binding affinity (K _D ) of 1 × 10 ^-8 or less than 1 × 10 ^-8 To dog TNF. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the binding of the antibody or antigen-binding fragment to canine TNF inhibits the ability of canine TNF to bind to the TNFR1 receptor. The antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the antigen-binding fragment is selected from the group consisting of a single-chain Fv (scFv) antibody fragment, a Fab antibody fragment, and a Fab' antibody fragment And F(ab') ₂ antibody fragments. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the antibody is a multivalent antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the antibody is a multispecific antibody. A method of treating, ameliorating or preventing an immune-mediated condition, The method comprises the steps of: providing a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 25, and administering it to a dog in need thereof. The method of claim 26, wherein the immune-mediated condition is a chronic inflammatory disease. The method of claim 27, wherein the chronic inflammatory disease is selected from the group consisting of immune-mediated polyarthritis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, Crohn Crohn's disease, ulcerative colitis, psoriasis and psoriatic arthritis, systemic vasculitis, atopic dermatitis, congestive heart failure, refractory uveitis, and bronchial asthma. The method of claim 26, wherein the immune-mediated condition is a TNF-α-related disorder selected from the group consisting of sepsis, septic shock, diabetes, Alzheimer's disease , Parkinson's disease, stroke, ischemic heart disease, and amyotrophic lateral sclerosis. The method of any one of claims 26 to 29, further comprising the step of co-administering at least one other therapeutic agent. The method of claim 30, wherein the at least one therapeutic agent is selected from the group consisting of methotrexate, canine TNF fusion protein, soluble p55 or p75 TNF receptor or a derivative thereof, for a canine Chimeric or canine antibodies to TNF, anti-canine TNF antibody fragments, analgesics, NSAIDs, opioids, corticosteroids, steroids, and neurogenics Long-factor antagonists. The method of claim 30, wherein the at least one therapeutic agent is selected from the group consisting of an antibiotic, an antifungal agent, an antiprotozoal agent, and an antiviral therapeutic agent. The method of claim 30, wherein the at least one therapeutic agent is selected from the group consisting of an inhibitor of an inflamma mediator, a PGE receptor antagonist, an immunosuppressive agent, cyclosporine, an anti-inflammatory sugar. Corticosteroids, agents for the treatment of cognitive dysfunction or cognitive disorders, antihypertensive agents, compounds for the treatment of cardiovascular dysfunction, diuretics, vasodilators, beta-adrenergic receptor antagonists, angiotensin- II Invertase inhibitor, calcium channel blocker and HMG-CoA reductase inhibitor. The method of any one of claims 26 to 33, wherein the antibody is administered to the dog at a dose ranging from about 0.01 mg/kg body weight to about 10 mg/kg body weight. A pharmaceutical composition for treating, ameliorating or preventing an immune-mediated condition, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, and at least one A pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutical composition according to claim 35, which additionally comprises at least one other therapeutic agent selected from the group consisting of methotrexate, canine TNF fusion protein, soluble p55 or p75 TNF receptor or a derivative thereof, Chimeric or canine antibodies against canine TNF, anti-canine TNF antibody fragments, analgesics, NSAIDs, opioids, corticosteroids, steroids, and nerve growth factor antagonists. An isolated nucleic acid encoding an antibody or antigen-binding fragment according to any one of claims 1 to 25. An isolated nucleic acid encoding a light chain variable domain of an anti-canine TNF canineized antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 1, or a light chain having the amino acid sequence of SEQ ID NO: . An isolated nucleic acid encoding a heavy chain variable domain of an anti-canine TNF canineized antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 2, or having selected from SEQ ID NO: 4, SEQ ID NO: 5 The heavy chain of the amino acid sequence of the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7. An isolated nucleic acid according to any one of claims 37 to 39, which is operably linked to one or more regulatory sequences. A performance vector comprising the nucleic acid according to any one of claims 37 to 39. An expression vector according to claim 41 of the patent application, which additionally comprises one or more regulatory sequences. The expression vector of claim 41 or 42, wherein the expression vector is a plastid or retroviral vector. A host cell which has the expression vector of any one of claims 41 to 43. A method of producing an anti-canine TNF neutralizing antibody, the method comprising the step of culturing a host cell as claimed in claim 44 to cause the cell to exhibit a canine anti-canine TNF neutralizing antibody. A method for treating, ameliorating or inhibiting an immune-mediated condition in a dog, the method comprising administering to the dog a therapeutically effective amount as claimed in the patent scope The step of the nucleic acid of any one of items 37 to 39. An antibody or antigen-binding fragment according to any one of claims 1 to 25, as in the pharmaceutical composition of claim 35 or 36, as in claim 37 to 39 A nucleic acid according to any one of claims 41 to 43 for use in the treatment or prevention of chronic inflammatory diseases in dogs. An antibody or antigen-binding fragment according to claim 47, wherein the chronic inflammatory disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis and other polyarthritis, immune-mediated multiple joints Inflammation, ankylosing spondylitis, Crohn's disease and ulcerative colitis, psoriasis and psoriatic arthritis, systemic vasculitis, atopic dermatitis, congestive heart failure, refractory uveitis, bronchial asthma and Allergic conditions. An antibody or antigen-binding fragment according to any one of claims 1 to 25, as in the pharmaceutical composition of claim 35 or 36, as in claim 37 to 39 A nucleic acid according to any one of claims 41 to 43 for use in the treatment of arthritis. An antibody or antigen-binding fragment according to claim 49, wherein the arthritis is selected from the group consisting of osteoarthritis, immune-mediated polyarthritis, and rheumatoid arthritis. An antibody or antigen-binding fragment according to any one of claims 1 to 25, wherein the pharmaceutical composition of claim 35 or 36, as claimed in claims 37 to 39 Any of them The use of a nucleic acid or a performance carrier according to any one of claims 41 to 43 for the preparation of a medicament for the treatment, amelioration or prevention of chronic inflammatory diseases in dogs. The use of an antibody or antigen-binding fragment according to claim 51, wherein the chronic inflammatory disease is selected from the group consisting of rheumatoid arthritis, immune-mediated polyarthritis, osteoarthritis and other multiple Arthritis, ankylosing spondylitis, Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis, systemic vasculitis, atopic dermatitis, congestive heart failure, refractory uveitis, bronchi Asthma and allergic conditions. An antibody or antibody-binding fragment according to any one of claims 1 to 25, wherein the pharmaceutical composition of claim 35 or 36, as claimed in claims 37 to 39 The use of a nucleic acid according to any one of claims 41 to 43 for the preparation of a medicament for the treatment, inhibition, amelioration or prevention of arthritis in dogs. The use of an antibody or antibody-binding fragment according to claim 53 wherein the arthritis is selected from the group consisting of osteoarthritis, immune-mediated polyarthritis, and rheumatoid arthritis. A cell line or a derivative or progeny cell thereof, which produces the anti-canine TNF neutralizing monoclonal antibody or fragment thereof according to any one of claims 1 to 25. A kit for treating a chronic inflammatory disease in a dog, comprising the anti-canine TNF antibody according to any one of claims 1 to 25 Its instruction manual.