Human Tumor Necrosis Factor Alpha Antibody

Abstract

The present disclosure relates to antibodies that specifically bind to soluble and membrane forms of human TNFα, compositions comprising such TNFα antibodies, and methods of using such TNFα antibodies and compositions.

Description

[Technical field]

[0001] Sequence Listing File This application has been submitted with a Sequence Listing in ST.26XML format. The Sequence Listing is provided as a file entitled "X30122_SequenceListing", created on September 20, 2022, and is 57 kilobytes in size. The Sequence Listing information in ST.26XML format is incorporated herein by reference in its entirety.

[0002] The present disclosure is in the field of medicine. In particular, the disclosure relates to antibodies that bind to soluble and membrane forms of human tumor necrosis factor alpha (TNFα), compositions comprising such TNFα antibodies, and methods of using such TNFα antibodies and compositions. [Background technology]

[0003] Chronic autoinflammatory immune disorders result from the body generating an immune response against its own tissues. Excessive and prolonged activation of immune cells such as T and B lymphocytes, and overexpression of the proinflammatory cytokine TNFα, along with other mediators such as interleukin-6 (IL-6), interleukin-1 (IL-1), and interferon gamma (IFN-γ), play a central role in the pathogenesis of autoinflammatory immune responses.

[0004] Tumor necrosis factor alpha (TNFα, also known as tumor necrosis factor, TNF, and cachectin) is a cytokine that activates macrophages, monocytes, and CD4 + and CD8 +TNFα is a pleiotropic homotrimeric cytokine reported to be secreted by T lymphocytes, natural killer (NK) cells, B cells, neutrophils, and endothelial cells. TNFα is expressed in both soluble and membrane forms (the membrane-bound precursor form can be proteolytically cleaved into soluble homotrimers by the metalloproteinase TNFα-converting enzyme (TACE)). Soluble TNFα (sTNFα) promotes various biological activities through type 1 receptors (also known as TNFR1, TNFRSF1A, CD120a, and p55) and type 2 receptors (also known as TNFR2, TNFRSF1B, CD120b, and p75). TNFα binds to its receptors, mainly TNFR1 and TNFR2, and transmits molecular signals for biological functions such as inflammation and cell death. TNFRs are activated by both sTNFα and transmembrane TNFα (tmTNFα). TNFα plays a role in regulating immune cells and is associated with chronic inflammation, especially acute phase inflammatory responses. Excessive amounts of TNFα are associated with a variety of chronic autoinflammatory immune disorders.

[0005] Anti-TNFα therapeutics targeting chronic autoinflammatory immune disorders are known and either approved or in clinical development. Such therapeutics include adalimumab, infliximab, golimumab, certolizumab, and etanercept. (Jang, Di., Int. J. Mol. Sci., 2021, 22(5):2719). However, a major drawback of their use is the generation of anti-drug antibodies in some patients receiving anti-TNFα therapeutics. Such anti-drug antibodies may be non-neutralizing antibodies that bind to the anti-TNFα therapeutic simultaneously with TNFα, or they may be neutralizing antibodies that reduce the effective concentration of the anti-TNFα therapeutic in serum and / or compete with TNFα for antigen binding sites (paratopes), thus inhibiting the mechanism of action of the anti-TNFα therapeutic. (Schie KA, et al, Annals of the Rheumatic Diseases, 2015, 74:311-314). For example, studies have shown that over 90 percent of anti-TNFα drug antibodies are neutralizing and may be cross-reactive with other anti-TNFα antibody therapeutics. (Schie KA, et al, 2015). Thus, in some cases, patients who develop anti-drug antibodies to anti-TNFα therapeutics have been reported to have reduced clinical responses to these therapeutics and / or adverse events such as infusion-related reactions characterized by symptoms such as fever, pruritus, bronchospasm, or cardiovascular collapse during or within the first day after drug administration (Atiqi, S., Front Immunol., 2020, 26(11):312). Thus, anti-drug antibody responses may provide patients with limited treatment options.

[0006] Thus, there remains a need for alternative anti-TNFα therapeutics that can neutralize soluble and membrane TNFα with the desired affinity, provide a sustained duration of action, and treat chronic autoinflammatory immune disorders. In particular, there remains a need for anti-human TNFα antibodies that have a reduced risk of eliciting anti-drug antibody responses and / or do not significantly bind anti-drug antibodies to other anti-TNFα antibody therapeutics. Furthermore, there remains a need for anti-human TNFα antibodies that can treat chronic autoinflammatory immune disorders and can treat chronic autoinflammatory immune disorders in patients who have developed anti-drug antibody responses to treatment with other TNFα therapeutics. Such anti-human TNFα antibodies also preferably have a good developability profile, such as low immunogenicity risk and / or good physicochemical properties for ease of development, manufacture, and formulation. Summary of the Invention

[0007] The present disclosure provides anti-human TNFα antibodies that bind and neutralize human TNFα and inhibit TNF receptor-mediated responses (e.g., NFkB activation, cytokine production). The present disclosure further provides compositions comprising such anti-human TNFα antibodies, as well as methods of using such anti-human TNFα antibodies and compositions thereof. In particular, the present disclosure provides anti-human TNFα antibodies that have desirable binding affinity, bind and neutralize soluble and membrane human TNFα, are internalized upon binding to membrane TNFα, and / or have low or no binding to anti-drug antibodies to other anti-TNFα therapeutics, and have potential use in treating patients with chronic autoinflammatory immune disorders who have developed anti-drug antibodies to previous treatment with an anti-TNFα therapeutic, such as adalimumab. Such chronic autoinflammatory immune disorders include rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), plaque psoriasis (PS), hidradenitis suppurativa (HS), uveitis, non-infectious intermediate uveitis, posterior uveitis or panuveitis, or Behcet's disease. The anti-human TNFα antibodies disclosed herein also preferably further exhibit a good developability profile, such as low immunogenicity risk and / or good physicochemical properties (e.g., viscosity, aggregation, stability) for ease of development, manufacture and formulation. Thus, the anti-human TNFα antibodies provided herein have one or more of the following properties: have a good developability profile, such as: 1) bind to human TNFα with a desired binding affinity; 2) bind to rhesus monkey and / or canine TNFα with a desired binding affinity; 3) inhibit TNFR-mediated signaling (e.g., NFkB); 4) inhibit cytokine production in vivo (e.g., CXCL1); 5) have low or no binding to other anti-TNFα therapeutics, e.g., anti-drug antibodies to adalimumab; 6) are internalized when bound to membrane TNFα; 7) have low immunogenicity risk; and / or 8) have an acceptable viscosity and / or aggregation profile for ease of development, manufacturing, and formulation. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows that an exemplary anti-human TNFα antibody Ab6 exhibits low or no binding to anti-drug antibodies against adalimumab generated in cynomolgus monkeys hyperimmunized with adalimumab. [Diagram 2] 1 shows that an exemplary anti-human TNFα antibody Ab6 exhibits low or no binding to anti-drug antibodies against adalimumab formed in human patients treated with adalimumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] In some embodiments, the disclosure provides an antibody that binds human TNFα and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, the antibody comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 9 and a light chain (LC) comprising SEQ ID NO: 10.

[0010] In some embodiments, the disclosure provides an antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; a. HCDR1 comprises SEQ ID NO: 22, HCDR2 comprises SEQ ID NO:23, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 4, 14, or 46; LCDR2 comprises SEQ ID NO:5, LCDR3 comprises SEQ ID NO:6, b. HCDR1 comprises SEQ ID NO: 22, HCDR2 comprises SEQ ID NO:23, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO:5, LCDR3 comprises SEQ ID NO: 15 or 47; c. HCDR1 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO: 30, LCDR1 comprises SEQ ID NO:31, LCDR2 comprises SEQ ID NO:5, LCDR3 comprises SEQ ID NO: 32; or d. HCDR1 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO:5, LCDR3 comprises SEQ ID NO:15.

[0011] In some embodiments, the disclosure provides an antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 13, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 15. In some embodiments, the antibody comprises a VH comprising SEQ ID NO: 16 and a VL comprising SEQ ID NO: 17. In some embodiments, the antibody comprises a HC comprising SEQ ID NO: 18 and a LC comprising SEQ ID NO: 19.

[0012] In some embodiments, the disclosure provides an antibody that binds human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO:22, HCDR2 comprising SEQ ID NO:23, HCDR3 comprising SEQ ID NO:13, LCDR1 comprising SEQ ID NO:4, LCDR2 comprising SEQ ID NO:5, and LCDR3 comprising SEQ ID NO:6. In some embodiments, the human TNFα antibody comprises a VH comprising SEQ ID NO:24 and a VL comprising SEQ ID NO:8. In some embodiments, the antibody that binds human TNFα comprises a heavy chain (HC) comprising SEQ ID NO:25 and a light chain (LC) comprising SEQ ID NO:10.

[0013] In some embodiments, the disclosure provides an antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 22, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 15. In some embodiments, the antibody comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 17. In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 25 and a light chain (LC) comprising SEQ ID NO: 19.

[0014] In some embodiments, the disclosure provides an antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 22, HCDR2 comprising SEQ ID NO: 23, HCDR3 comprising SEQ ID NO: 13, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6. In some embodiments, the antibody comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 27. In some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 25 and a light chain (LC) comprising SEQ ID NO: 28.

[0015] In some embodiments, the disclosure provides an antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO:22, HCDR2 comprising SEQ ID NO:23, HCDR3 comprising SEQ ID NO:13, LCDR1 comprising SEQ ID NO:46, LCDR2 comprising SEQ ID NO:5, and LCDR3 comprising SEQ ID NO:6. In some embodiments, SEQ ID NO:46 comprises amino acid residue QASQGIXaa7NYLN, and Xaa7 of SEQ ID NO:46 is serine or arginine. In some embodiments, the antibody comprises a VH comprising SEQ ID NO:24 and a VL comprising SEQ ID NO:8. In some embodiments, the antibody comprises a VH comprising SEQ ID NO:24 and a VL comprising SEQ ID NO:27. In some embodiments, the antibody comprises a heavy chain (HC) that comprises SEQ ID NO: 25 and a light chain (LC) that comprises SEQ ID NO: 10. In some embodiments, the antibody comprises a heavy chain (HC) that comprises SEQ ID NO: 25 and a light chain (LC) that comprises SEQ ID NO: 28.

[0016] In some embodiments, the disclosure provides an antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO:22, HCDR2 comprising SEQ ID NO:23, HCDR3 comprising SEQ ID NO:13, LCDR1 comprising SEQ ID NO:14, LCDR2 comprising SEQ ID NO:5, and LCDR3 comprising SEQ ID NO:47. In some embodiments, SEQ ID NO:47 comprises amino acid residue QQYDXaa5LPLT, and Xaa5 of SEQ ID NO:47 is asparagine or lysine. In some embodiments, the antibody comprises a VH comprising SEQ ID NO:24 and a VL comprising SEQ ID NO:17. In some embodiments, the antibody comprises a VH comprising SEQ ID NO:24 and a VL comprising SEQ ID NO:27. In some embodiments, the antibody comprises a heavy chain (HC) that comprises SEQ ID NO: 25 and a light chain (LC) that comprises SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain (HC) that comprises SEQ ID NO: 25 and a light chain (LC) that comprises SEQ ID NO: 28.

[0017] In some embodiments, the disclosure provides an antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 30, LCDR1 comprising SEQ ID NO: 31, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 32. In some embodiments, the antibody comprises a VH comprising SEQ ID NO: 33 and a VL comprising SEQ ID NO: 34. In some embodiments, the antibody that binds to human TNFα comprises a heavy chain (HC) comprising SEQ ID NO: 35 and a light chain (LC) comprising SEQ ID NO: 36.

[0018] In some embodiments of the present disclosure, the anti-human TNFα antibody is a fully human antibody. In some embodiments of the present disclosure, the anti-human TNFα antibody has a human IgG1 isotype.

[0019] In some embodiments of the disclosure, the anti-human TNFα antibody comprises a modified human IgG1. In some embodiments, the modification is in the heavy chain variable region (VH). In some embodiments, the modification is in the light chain variable region (VL). In some embodiments, the modification is in the VH and VL. In further embodiments, the modified human IgG1 VH and / or VL provides the anti-human TNFα antibody of the disclosure with a desirable viscosity profile and / or immunogenicity risk profile.

[0020] In further embodiments of the disclosure, the anti-human TNFα antibody has a modified human IgG1 constant domain that includes an engineered cysteine ​​residue for use in generating antibody conjugate compounds (also referred to as bioconjugates) (see WO 2018 / 232088(A1)). More particularly, in such embodiments of the disclosure, the anti-human TNFα antibody includes a cysteine ​​at amino acid residue 124 (EU numbering), or a cysteine ​​at amino acid residue 378 (EU numbering), or a cysteine ​​at amino acid residue 124 (EU numbering) and at amino acid residue 378 (EU numbering). Also provided herein are antibody drug conjugates that include the anti-human TNFα antibodies disclosed herein.

[0021] In some embodiments of the present disclosure, the anti-human TNFα antibodies bind to soluble and membrane TNFα and inhibit TNFα binding to human TNF receptors (TNFRs). In some embodiments of the present disclosure, the anti-human TNFα antibodies bind to soluble and membrane human TNFα and inhibit human TNFα binding to human TNF receptors and inhibit TNFR-mediated responses. In some embodiments, the anti-human TNFα antibodies of the present disclosure inhibit human TNFα binding to human TNFR and thus inhibit TNFR-mediated responses such as human TNFR activation, NFkB phosphorylation, cytokine production, and / or soluble and membrane TNFα-induced cell death. In some embodiments, the anti-human TNFα antibodies of the present disclosure bind to human TNFα and inhibit TNFR-mediated NFkB phosphorylation and signaling on TNFR-expressing cells by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In further embodiments, the anti-human TNFα antibodies of the present disclosure bind to human TNFα and inhibit TNFα-induced cytokine production (e.g., CXCL1) by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In yet further embodiments, the anti-human TNFα antibodies of the present disclosure bind to human TNFα and inhibit TNFα-induced cytokine production (e.g., CXCL1) by about 45% to about 95%. In further embodiments, the anti-human TNFα antibodies of the present disclosure bind to soluble human TNFα and inhibit TNFα-induced cell death by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In yet other embodiments, the anti-human TNFα antibodies of the present disclosure bind to membrane TNFα and inhibit membrane TNFα-induced cell death by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0022] In some embodiments, the anti-human TNFα antibodies of the present disclosure bind to membrane human TNFα and are internalized by membrane TNFα-expressing cells. In such embodiments, the antibodies of the present disclosure bind to membrane human TNFα and are about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% internalized by membrane TNFα-expressing cells.

[0023] In some embodiments, the anti-human TNFα antibodies of the present disclosure exhibit low or no binding to anti-drug antibodies against other anti-TNFα therapeutics (e.g., adalimumab, infliximab, golimumab, certolizumab, or etanercept). In certain embodiments, the anti-human TNFα antibodies of the present disclosure exhibit low or no binding to anti-drug antibodies against adalimumab. In such embodiments, the anti-human TNFα antibodies of the present disclosure may be used to treat patients who have developed anti-drug antibodies against prior treatment with other anti-TNFα therapeutics as defined herein (e.g., adalimumab). In further embodiments, the anti-human TNFα antibodies of the present disclosure may be used to treat patients who have developed anti-drug antibodies against such other anti-TNFα therapeutics from prior treatment with other anti-TNFα therapeutics, and thus have a reduced clinical response or adverse reaction to the other anti-TNFα therapeutics. In such embodiments, the anti-human TNFα antibodies of the disclosure have sufficiently different amino acid and nucleic acid sequences that they have low or no binding to anti-drug antibodies to other anti-TNFα therapeutics. In certain embodiments, the anti-human TNFα antibodies of the disclosure have sufficiently different CDR amino acid sequences that they have low or no binding to anti-drug antibodies to other anti-TNFα therapeutics. In some embodiments, the other anti-TNFα therapeutic is adalimumab, infliximab, golimumab, certolizumab, or etanercept.

[0024] In some embodiments, the disclosure provides a nucleic acid encoding the HC or LC, or the VH or VL, of a novel antibody that binds human TNFα, or a vector comprising such a nucleic acid.

[0025] In some embodiments, the disclosure provides a nucleic acid comprising the sequence of SEQ ID NO: 11, 12, 20, 21, 26, 29, 37, or 38.

[0026] In some embodiments, a nucleic acid is provided that encodes a heavy or light chain of an antibody that binds to human TNFα. In some embodiments, a nucleic acid is provided that comprises a sequence encoding SEQ ID NO: 9, 10, 18, 19, 25, 28, 35, or 36. In some embodiments, a nucleic acid is provided that comprises a sequence encoding an antibody heavy chain that comprises SEQ ID NO: 9, 18, 25, or 35. For example, the nucleic acid can comprise a sequence selected from SEQ ID NO: 11, 20, 26, or 37. In some embodiments, a nucleic acid is provided that comprises a sequence encoding an antibody light chain that comprises SEQ ID NO: 10, 19, 28, or 36. For example, the nucleic acid can comprise a sequence selected from SEQ ID NO: 12, 21, or 29, or 38.

[0027] In some embodiments of the present disclosure, a nucleic acid is provided that encodes a VH or VL of an antibody that binds human TNFα. In some embodiments, a nucleic acid is provided that comprises a sequence encoding SEQ ID NO: 7, 8, 16, 17, 24, 27, 33, or 34. In some embodiments, a nucleic acid is provided that comprises a sequence encoding an antibody VH that comprises SEQ ID NO: 7, 16, 24, or 33. In some embodiments, a nucleic acid is provided that comprises a sequence encoding an antibody VL that comprises SEQ ID NO: 8, 17, 27, or 34.

[0028] In some embodiments of the present disclosure, a vector is provided that comprises a nucleic acid sequence encoding an antibody heavy or light chain. For example, such a vector can comprise a nucleic acid sequence encoding SEQ ID NO: 9, 18, 25, or 35. In some embodiments, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 10, 19, 28, or 36.

[0029] Also provided herein is a vector comprising a nucleic acid sequence encoding an antibody VH or VL. For example, such a vector can comprise a nucleic acid sequence encoding SEQ ID NO: 7, 16, 24, or 33. In some embodiments, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 8, 17, 27, or 34.

[0030] Also provided herein is a vector comprising a first nucleic acid sequence encoding an antibody heavy chain and a second nucleic acid sequence encoding an antibody light chain. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 9, 18, 25, or 35, and a second nucleic acid sequence encoding SEQ ID NO: 10, 19, 28, or 36.

[0031] In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:9 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:18 and a second nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:25 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:25 and a second nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:25 and a second nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:25 and a second nucleic acid sequence encoding SEQ ID NO:28. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:35 and a second nucleic acid sequence encoding SEQ ID NO:36.

[0032] Also provided herein are compositions comprising a first vector comprising a nucleic acid sequence encoding an antibody heavy chain and a second vector comprising a nucleic acid sequence encoding an antibody light chain, in some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9, 18, 25, or 35 and a second nucleic acid sequence encoding SEQ ID NO: 10, 19, 28, or 36.

[0033] In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:9 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:18 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:28. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:35 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:36.

[0034] The nucleic acid of the present disclosure can be expressed in a host cell, for example, after the nucleic acid is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked nucleic acid are well known in the art. The expression vector can include a sequence encoding one or more signal peptides that facilitate secretion of a polypeptide from a host cell. The expression vector containing a nucleic acid of interest (e.g., a nucleic acid encoding an antibody heavy or light chain) can be transferred to a host cell by well-known methods, for example, stable or transient transfection, transformation, transduction or infection. Additionally, the expression vector can include one or more selection markers, such as, for example, tetracycline, neomycin, and dihydrofolate reductase, to aid in the detection of host cells transformed with the desired nucleic acid sequence.

[0035] In another aspect, provided herein is a cell, e.g., a host cell, comprising a nucleic acid, vector, or nucleic acid composition described herein. The host cell may be a cell stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or a portion of the antibodies described herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with an expression vector expressing the HC and LC polypeptides of the antibodies of the present disclosure. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing the HC polypeptide and a second vector expressing the LC polypeptide of the antibodies described herein. Such a host cell, e.g., a mammalian host cell, may express an antibody that binds human TNFα as described herein. Mammalian host cells known to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NS0 cells.

[0036] In some embodiments, a cell, e.g., a host cell, comprises a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 9, 18, 25, or 35 and a second nucleic acid sequence encoding SEQ ID NO: 10, 19, 28, or 36.

[0037] In some embodiments, a cell, e.g., a host cell, comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 9, 18, 25, or 35, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 10, 19, 28, or 36.

[0038] The disclosure further provides a method for producing an antibody that binds human TNFα as described herein by culturing the above-described host cells, e.g., mammalian host cells, under conditions in which the antibody is expressed and recovering the expressed antibody from the culture medium. The medium into which the antibody is secreted can be purified by conventional techniques. Various methods of protein purification can be employed, such methods being known in the art and described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0039] The disclosure further provides an antibody, or antigen-binding fragment thereof, produced by any of the methods described herein.

[0040] In another aspect, provided herein is a pharmaceutical composition comprising the antibody, nucleic acid, or vector described herein.Such pharmaceutical composition may also comprise one or more pharma- ceutical acceptable excipients, diluents, or carriers.The pharmaceutical composition can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press).

[0041] The antibodies, nucleic acids, vectors, or pharmaceutical compositions that bind human TNFα described herein may be used to treat a TNFα-associated disorder (e.g., a chronic autoinflammatory immune disorder, including, but not limited to, rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, or Behcet's disease).

[0042] In some embodiments, provided herein is a method of treating a TNFα-related disorder, e.g., a chronic autoinflammatory immune disorder, in a subject (e.g., a human patient) in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody that binds human TNFα, a nucleic acid encoding such an antibody that binds human TNFα, a vector comprising such a nucleic acid, or a pharmaceutical composition comprising such an antibody, nucleic acid, or vector that binds human TNFα as described herein. The antibody, nucleic acid, vector, or pharmaceutical composition described herein can be administered by a parenteral route (e.g., subcutaneously and intravenously). In embodiments, the TNFα-related disorder is a chronic autoinflammatory immune disorder. Such chronic autoinflammatory immune disorders include, but are not limited to, rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, or Behcet's disease. In some embodiments, the subject receiving a therapeutically effective amount of an antibody that binds human TNFα has been pretreated with another anti-TNFα therapeutic, and the subject has generated anti-drug antibodies against the other anti-TNFα therapeutic. In such embodiments, the other anti-TNFα therapeutic is selected from adalimumab, infliximab, golimumab, certolizumab, or etanercept. In yet further embodiments, the anti-human TNFα antibody as disclosed herein has low or no binding to anti-drug antibodies against at least four or more of the other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept. In yet further embodiments, the anti-human TNFα antibody as disclosed herein has low or no binding to anti-drug antibodies against at least three or more of the other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept.In still further embodiments, the anti-human TNFα antibodies as disclosed herein exhibit low or no binding to anti-drug antibodies against at least two or more other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept. In yet other embodiments, the anti-human TNFα antibodies disclosed herein exhibit low or no binding to anti-drug antibodies against adalimumab.

[0043] Also provided herein is an antibody, nucleic acid, vector, or pharmaceutical composition that binds human TNFα described herein for use in therapy. Additionally, the present disclosure also provides an antibody, nucleic acid, vector, or pharmaceutical composition that binds human TNFα described herein for use in the treatment of TNFα-related disorders, such as chronic autoinflammatory immune disorders. Such chronic autoinflammatory immune disorders include, but are not limited to, rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, or Behcet's disease. The antibody, nucleic acid, vector, or pharmaceutical composition described herein can be administered by parenteral routes (e.g., subcutaneous and intravenous). In some embodiments of the present disclosure, a subject receiving a therapeutically effective amount of an antibody that binds human TNFα has been pretreated with another anti-TNFα therapeutic, and the subject has generated anti-drug antibodies against the other anti-TNFα therapeutic. In such embodiments, the other anti-TNFα therapeutic is selected from adalimumab, infliximab, golimumab, certolizumab, or etanercept. In yet further embodiments, an anti-human TNFα antibody as disclosed herein has low or no binding to anti-drug antibodies against at least four or more of the other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept. In yet further embodiments, an anti-human TNFα antibody as disclosed herein has low or no binding to anti-drug antibodies against at least three or more of the other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept. In still further embodiments, the anti-human TNFα antibodies as disclosed herein exhibit low or no binding to anti-drug antibodies to at least two or more other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept.In yet other embodiments, the anti-human TNFα antibodies disclosed herein exhibit low or no binding to anti-drug antibodies directed against adalimumab.

[0044] Provided herein is the use of an antibody, nucleic acid, vector, or pharmaceutical composition that binds human TNFα described herein in the manufacture of a medicament for the treatment of a TNFα-related disorder, such as a chronic autoinflammatory immune disorder, including, but not limited to, rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, or Behcet's disease.

[0045] In some embodiments, the antibodies of the present disclosure bind human TNFα and have low or no binding to anti-drug antibodies against other anti-TNFα therapeutics. In such embodiments, the anti-human TNFα antibodies of the present disclosure bind human TNFα, neutralize soluble and membrane human TNFα, and inhibit TNF receptor-mediated responses. In some embodiments, the other anti-TNFα therapeutics are selected from adalimumab, infliximab, golimumab, certolizumab, or etanercept. In some embodiments, the anti-human TNFα antibodies of the present disclosure have low or no binding to anti-drug antibodies against at least four or more of the other anti-TNFα therapeutics consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept. In other embodiments, the anti-human TNFα antibodies of the present disclosure bind poorly or do not bind to anti-drug antibodies against at least three or more other anti-TNFα therapeutics consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept. In other embodiments, the anti-human TNFα antibodies of the present disclosure bind poorly or do not bind to anti-drug antibodies against at least two or more other anti-TNFα therapeutics consisting of adalimumab, infliximab, golimumab, certolizumab, or etanercept. In other embodiments, the anti-human TNFα antibodies of the present disclosure bind poorly or do not bind to anti-drug antibodies against adalimumab. In such embodiments, the anti-human TNFα antibodies of the present disclosure are IgG1. In further embodiments, the anti-human TNFα antibodies of the present disclosure bind to human TNFα and have low or no binding to anti-drug antibodies to other anti-TNFα therapeutics, and the anti-human TNFα antibodies comprise a heavy chain (HC) and a light chain (LC), where the HC comprises SEQ ID NO: 9, 18, 25, or 35, and the LC comprises SEQ ID NO: 10, 19, 28, or 36. In such embodiments, the anti-human TNFα antibodies of the present disclosure neutralize human TNFα and inhibit TNF receptor-mediated responses. In further embodiments, the anti-human TNFα antibodies of the present disclosure are internalizing antibodies. In yet further embodiments, the anti-human TNFα antibodies of the present disclosure have low immunogenicity.

[0046] The term "TNFα" as used herein refers to soluble and membrane TNFα, as well as any naturally occurring mature TNFα resulting from processing of intracellular TNFα precursor protein, unless otherwise stated. The term includes TNFα from any vertebrate source, including mammals such as dogs, primates (e.g., humans and cynomolgus or rhesus monkeys), and rodents (e.g., mice and rats), unless otherwise stated. The term also includes naturally occurring variants of TNFα, such as splice variants or allelic variants. An exemplary amino acid sequence of human TNFα is known in the art, e.g., NCBI accession number NP_000585 (SEQ ID NO: 39). An exemplary amino acid sequence of cynomolgus TNFα is also known in the art, e.g., UniProt reference sequence P79337 (SEQ ID NO: 45). The amino acid sequence of an example of rhesus monkey TNFα is also known in the art, e.g., UniProt reference sequence P48094 (SEQ ID NO: 40). The amino acid sequence of an example of canine TNFα is also known in the art, e.g., GenBank Accession No. CAA64403 (SEQ ID NO: 44). The term human "TNFα" is used herein to collectively refer to all known human TNFα isoforms and polymorphic forms. Sequencing as used herein is based on the mature protein without the signal peptide.

[0047] The term "TNFR" or "TNF receptor" as used herein refers to any naturally occurring mature TNFR, such as TNFR1 (also known as p55 or p60) or TNFR2 (also known as p75 or p80), unless otherwise indicated. The term includes TNFR from any vertebrate source, including mammals such as dogs, primates (e.g., humans and cynomolgus or rhesus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of TNFR, such as splice variants or allelic variants. An exemplary amino acid sequence of human TNFR1 is known in the art, e.g., GenBank Accession No. AAA61201 (SEQ ID NO: 48). An exemplary amino acid sequence of human TNFR2 is known in the art, e.g., NCBI Accession No. NP_001057 (SEQ ID NO: 49). The term "TNFR" is used herein to collectively refer to all known human TNFR isoforms and polymorphic forms.

[0048] As used herein, the term "TNFα-related disorder" refers to a disorder associated with dysregulation of TNFα-induced TNF receptor-mediated signaling, such as a disorder associated with dysregulation of TNFα-induced TNFR1 and / or TNFR2 signaling. Such TNFα-mediated disorders may include, for example, chronic autoinflammatory immune disorders as disclosed herein.

[0049] The term "anti-drug antibody" or "anti-drug antibody, ADA" as used herein refers to an antibody formed in a mammal from an immune response to a therapeutic agent administered to the mammal. In some embodiments of the present disclosure, anti-drug antibodies formed against a therapeutic agent may neutralize the effect of the therapeutic agent, thus altering the pharmacokinetic (PK) and / or pharmacodynamic (PD) properties of the therapeutic agent, interfering with the effect of the therapeutic agent and / or reducing the efficacy and / or reducing the clinical response to the therapeutic agent. Anti-drug antibodies against a therapeutic agent may also lead to adverse immune reactions in the patient, such that the patient may not be a candidate for further treatment with the therapeutic agent. Examples of adverse immune reactions include, but are not limited to, infusion-related reactions characterized by symptoms such as fever, pruritus, bronchospasm, or cardiovascular collapse on the first day after drug administration (Atiqi, S., Front Immunol., 2020, 26(11):312).

[0050] As used herein, the term "low or no binding" to an anti-drug antibody refers to binding of an anti-human TNFα antibody of the present disclosure to an anti-drug antibody to another anti-TNFα therapeutic agent, where such binding is determined to be below the cutoff point of the assay used to measure binding or within a predetermined variability range of the assay. In such methods, the cutoff point is a predetermined threshold used to identify positive binding to an anti-drug antibody. In some embodiments, the predetermined variability of the assay is less than about 20% above the cutoff point of the assay. In such embodiments, binding of an anti-human TNFα antibody of the present disclosure to an anti-drug antibody to another therapeutic agent (e.g., adalimumab) that is less than about 20% above the cutoff point of the assay is considered low binding. In some embodiments, binding of an anti-human TNFα antibody of the present disclosure to an anti-drug antibody to another therapeutic agent (e.g., adalimumab) that is equal to or below the cutoff point of the assay is considered no binding.

[0051] The term "other anti-TNFα therapeutic agent" refers to an agent that binds to TNFα and inhibits TNF receptor-mediated responses, and does not include the anti-human TNFα antibodies described herein. Such agents include antibodies, antibody fragments, or antigen-binding fragments that include at least a portion of an antibody that retains the ability to interact with an antigen, such as, but not limited to, a Fab, Fab', F(ab')2, Fv fragment, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragment, or linear antibodies, which may be fused to an Fc region or an IgG heavy chain constant region. In some embodiments, the other anti-TNFα therapeutic agent may be, for example, adalimumab, infliximab, golimumab, and certolizumab, and / or etanercept.

[0052] The term "antibody" as used herein refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. Antibodies may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments, and the term "antibody fragment or antigen-binding fragment" includes at least a portion of an antibody that retains the ability to interact with an antigen, such as Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragments, and linear antibodies, which may be fused to an Fc region or an IgG heavy chain constant region.

[0053] An exemplary antibody is an immunoglobulin G (IgG) type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino terminal portion of each of the four polypeptide chains contains a variable region of about 100-125 or more amino acids that is primarily responsible for antigen recognition. The carboxy terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region refers to the region of an antibody that includes the Fc region and the CH1 domain of the antibody heavy chain. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The IgG isotype can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). The numbering of amino acid residues in the constant regions is based on the EU index as in Kabat. Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: USDept. of Health and Human Services, Public Health Service, National Institutes of Health (1991). The terms EU index numbering or EU numbering are used interchangeably herein.

[0054] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). The CDRs are exposed on the surface of the protein and are the regions of the antibody that are important for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3" and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3". The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be performed using the methods described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database, available at www.imgt.org; Lefranc et al., Nucleic Acids Res. 1999;27:209-212). A combination of the IMGT and North CDR definitions was used for the exemplary anti-human TNFα antibodies as described herein.

[0055] The term "Fc region" as used herein refers to the region of an antibody that includes the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may include a portion of the hinge region or the entire hinge region of the antibody heavy chain. Biological activities, such as effector functions, are attributed to the Fc region, which vary depending on the antibody isotype. Examples of antibody effector functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, complement-dependent cytotoxicity (CDC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0056] The term "epitope" as used herein refers to the amino acid residues of an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope. The term "epitope" can be used in reference to a structural epitope. A structural epitope can be used according to some embodiments to describe the area of ​​an antigen that is covered by an antibody (e.g., the footprint of an antibody when bound to the antigen). In some embodiments, a structural epitope can describe amino acid residues of an antigen that are within a certain proximity (e.g., within a certain number of angstroms) of amino acid residues of an antibody. The term "epitope" can also be used in reference to a functional epitope. A functional epitope can be used according to some embodiments to describe amino acid residues of an antigen that interact with amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody. Epitopes can be determined according to different experimental techniques, also called "epitope mapping techniques". It is understood that the determination of an epitope may vary based on the different epitope mapping techniques used, and may also vary with different experimental conditions used, for example, due to conformational changes or cleavage of the antigen induced by the particular experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3 rd ed. 2018; Holst et al., Molecular Pharmacology 1998, 53(1):166-175), including, but not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species-exchange mutagenesis, alanine scanning mutagenesis, steric hindrance mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

[0057] The term "bind," as used herein, unless otherwise specified, is intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in the proximity of the two proteins or molecules, as determined by common methods known in the art.

[0058] The term "nucleic acid" as used herein refers to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs. A polynucleotide of the disclosure can also include substrates incorporated therein, for example, by a DNA or RNA polymerase or a synthetic reaction.

[0059] As used herein, the term "subject" refers to a mammal, including, but not limited to, humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Preferably, the subject is a human.

[0060] The term "therapeutically effective amount" as used herein refers to an amount of a protein or nucleic acid or vector or composition that induces a desired biological or medical response in a subject, such as reducing or inhibiting the activity of a protein, or improves symptoms, alleviates a condition, slows or delays the progression of a disease, or prevents a disease, etc. In one non-limiting embodiment, the term "therapeutically effective amount" refers to the necessary amount (dosage and duration and means of administration) of a protein or nucleic acid or vector or composition that, when administered to a subject, is effective to at least partially alleviate, inhibit, prevent and / or ameliorate a condition, or disorder or disease, to achieve a desired therapeutic result. A therapeutically effective amount of a protein or nucleic acid or vector or composition may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein or nucleic acid or vector or composition to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the protein or nucleic acid or vector or composition of the present disclosure are outweighed by the therapeutically beneficial effects.

[0061] The term "inhibit" as used herein refers to, for example, decreasing, reducing, slowing, diminishing, ceasing, disrupting, eliminating, antagonizing, or blocking a biological response or activity, but does not necessarily indicate a complete elimination of the biological response.

[0062] As used herein, "treatment" or "treating" refers to any process that may slow, control, retard, or halt the progression of a disorder or disease symptom disclosed herein, but does not necessarily indicate the complete disappearance of all disorder or disease symptoms. Treatment includes the administration of a protein or nucleic acid or vector or composition for the treatment of a disease or condition in a patient, particularly a human.

[0063] The term "neutralize" as used herein refers to the ability of an antibody, antibody fragment, or binding molecule to oppose, inactivate or neutralize at least one activity or function of an antigen.

[0064] As used herein, the term "about" means within 10%.

[0065] As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) are to be construed to encompass both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0066] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows that an exemplary anti-human TNFα antibody Ab6 exhibits low or no binding to anti-drug antibodies against adalimumab generated in cynomolgus monkeys hyperimmunized with adalimumab. FIG. 2 shows that an exemplary anti-human TNFα antibody Ab6 exhibits low or no binding to anti-drug antibodies against adalimumab formed in human patients treated with adalimumab. EXAMPLES

[0067] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0068] Example 1: Generation and engineering of anti-human TNFα antibodies Antibody generation: To generate antibodies specific for human TNFα, transgenic mice carrying human immunoglobulin variable regions were immunized with recombinant human TNFα. Screening was performed with human TNFα to test for cross-reactivity with other TNFα species. Antibodies cross-reactive with cynomolgus TNFα were cloned, expressed, purified by standard procedures, and tested for neutralization in a TNFα-induced cytotoxicity assay. Antibodies were selected and their CDRs, variable domain framework regions, and IgG isotype were engineered to improve binding affinity and developability properties such as stability, solubility, viscosity, hydrophobicity, and aggregation.

[0069] The amino acid sequence of human TNFα is provided as SEQ ID NO:39, and the amino acid sequence of cynomolgus TNFα is provided as SEQ ID NO:45.

[0070] TNFα antibodies can be synthesized and purified by well-known methods. Suitable host cells, such as Chinese Hamster Ovary cells (CHO), can be transiently or stably transfected with an expression system to secrete antibodies using a defined HC:LC vector ratio, when two vectors are used, or using a single vector system encoding both the heavy and light chains. The clarified medium into which the antibodies are secreted can be purified using commonly used techniques.

[0071] Antibody engineering to improve viscosity: The parent TNFα antibody line was found to have high viscosity when concentrated. Viscosity is an important developability criterion for evaluating the feasibility of delivering therapeutic antibodies via an autoinjector. Mutagenesis analysis of the antibodies required a delicate balance between improving biophysical properties and retaining the desired affinity and potency without increasing the risk of immunogenicity. In silico modeling of the parent antibody was used to identify regions of charge imbalance at the surface consisting of six complementarity determining regions (CDRs). Antibodies generated from mutagenesis were screened for TNFα binding, and those antibodies that retained or improved target binding compared to the parent mAb (as determined by ELISA) and had the desired viscosity and other developability properties were selected for further development.

[0072] Antibody engineering to reduce immunogenicity risk: Exemplary anti-human TNFα antibodies were further tested in MHC-associated peptide proteomics (MAPPS) assays to determine immunogenicity risk. Briefly, major histocompatibility complex (MHC) binding peptides were identified for antibodies with specific CDR sequences. CDR libraries with mutations identified as likely to reduce immunogenicity were constructed and screened for TNFα binding. Antibodies were screened and selected to engineer and optimize for low immunogenicity risk while balancing the desired binding affinity to TNFα and maintaining other desirable developability properties.

[0073] Tables 1 and 2 show exemplary anti-human TNFα antibody sequences that have been engineered to balance low viscosity, low immunogenicity risk, and other desirable developability properties while retaining desirable binding affinity to human TNFα.

[0074] [Table 1]

[0075] [Table 2]

[0076] Example 2. Binding Affinities of Exemplary Anti-Human TNFα Antibodies Binding affinity, method 1: The binding affinity of antibodies to human, rhesus monkey, mouse, rat, rabbit, and dog TNFα proteins was tested in an antigen-down ELISA format. Briefly, 384-well high binding plates (Greiner Bio-one #781061) were coated with 20 μL / well of 1 μg / mL human TNFα (Syngene), 2 μg / mL rhesus TNFα (R&D Systems, Catalog No. 1070-RM), 2 μg / mL mouse TNFα (R&D Systems, Catalog No. 410-MT / CF), or 2 μg / mL rat TNFα (R&D Systems, Catalog No. 510-RT-CF), 2 μg / mL rabbit TNFα (R&D Systems, Catalog No. 5670-TG / CF), or 2 μg / mL dog TNFα (R&D Systems, Catalog No. 1507-CT / CF) diluted in carbonate buffer pH 9.3 (0.015 M Na2CO3 and 0.035 M NaHCO3) and stored at 4° C. overnight. The next day, the plate was blocked with 80 μL of casein (Thermo Fisher Pierce, Cat. No. 37528) for 1 hour at room temperature, the blocking buffer was removed, and 20 μL of titrated purified antibody expressed in CHO cells (starting concentration at 20 μg / mL diluted in casein, titrated 3-fold, 8 points down) was added to the plate. The plate was incubated at 37°C for 90 minutes and then washed 3 times in PBS / 0.1% Tween. 20 μL of secondary antibody reagent goat anti-human kappa-AP (Southern Biotech, Cat. No. 2060-04) at 1:1500 dilution was added to the plate and incubated at 37°C for 45 minutes. The plate was washed 3 times in PBS / 0.1% Tween and 20 μL of alkaline phosphatase substrate solution diluted 1:35 in molecular grade water was added to each well. Once color had developed (approximately 15-30 min), plates were read at 560 nM OD on a Molecular Device Spectramax plate reader and data were acquired using Softmax Pro 4.7 software. Data analysis was performed in GraphPad Prism.

[0077] The results, shown in Table 3, demonstrate that exemplary anti-human TNFα antibodies Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 bind to human, rhesus, and canine TNFα with desirable affinities.

[0078] [Table 3]

[0079] Binding affinity, method 2: An MSD Sector S 600 instrument (Meso Scale Discovery, Rockville, MD) was used to read the MSD plates. Human and cynomolgus TNFα were biotinylated using Thermo-Fisher biotinylation kits. MSD assay plates were prepared as follows: Multi-array streptavidin-coated 96-well plates (Meso Scale Discovery, Cat# L15SA-1) were coated with 40 μL / well of a 1 μg / mL solution of either biotinylated human TNFα or biotinylated cynomolgus TNFα in PBS for 1 hour at room temperature (approximately 25° C.). After coating, the plates were washed three times in PBS+0.1% Tween (PBST) and then blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. The plates were then washed three times with PBS before adding the sample solution. Solution equilibrium titration (SET) samples were prepared in 1% BSA. Anti-human TNFα antibody Ab1 was diluted to 10 pM and TNFα was serially diluted for a total of 12 dilutions. The TNFα titration and immobilized antibody solutions were combined 1:1 to prepare the SET solution. The SET solution was incubated at 37°C for approximately 72 hours to allow binding to reach equilibrium. 40 μl of the SET solution was transferred to triplicate rows of the prepared MSD plate, incubated at room temperature for 2.5 minutes, and agitated by gently tapping the plate manually to capture free antibody. After incubation, the plate was washed 3 times with PBST. 40 μL of 1 μg / mL SULFO-Tag anti-human / NHP kappa antibody (Meso Discovery Scale, Cat# D20TF-6) in 1% BSA was then added to all wells. This was incubated statically at room temperature for 1 hour. The plate was then washed three times with PBST and then read after adding 150 μL of MSD GOLD Read Buffer A (Meso Scale Discovery, Cat. No. R92TG-2). The dilution series was prepared in triplicate in each individual experiment and three independent replicates were performed. D was determined from the MSD-SET data using a second-order kinetics model in XLfit.D Values ​​were entered into GraphPad Prism for both human and cynomolgus TNFα to determine standard deviation statistics.

[0080] Results from this assay show that the exemplary anti-human TNFα antibody Ab1 has a K D It binds to human TNFα with a K of 21.2 ± 7.4 pM D We show that it binds to cynomolgus monkey TNFα.

[0081] Example 3: Functional activity of anti-human TNFα antibodies Membrane-bound internalizing TNFα antibodies: Internalization of an exemplary anti-human TNFα antibody upon binding to membrane-expressed TNFα was tested in vitro in a CHO cell line stably transfected to express membrane human TNFα (uncleaved TNFα). Briefly, F(ab')2 fragment goat anti-human IgG (Jackson#109-006-098) was conjugated to pHrodo pH-sensitive dye (Fisher P36014) using the manufacturer's protocol. An exemplary anti-human TNFα antibody was incubated with an equimolar amount of F(ab')2 goat anti-hIgG-pHrodo in CHO growth medium at room temperature for 30 minutes. The antibody-dye mixture was added to CHO human TNFα transfected cells and then incubated at 37°C in a 5% CO2 shaker incubator for 3, 6 and 24 hours. Final concentrations of the exemplary TNFα antibodies were 10 μg / mL and 3.3 μg / mL. Cells were washed at the indicated time points and analyzed on a BD Fortessa flow cytometer.

[0082] The results shown in Table 4 indicate that the tested anti-human TNFα antibodies were internalized into CHO cells when bound to membrane human TNFα expressed on CHO cells at 3.33 μg / mL and 10 μg / mL. The results further indicated that the anti-human TNFα antibodies were internalized into lysosomes when bound to membrane TNFα expressed on lysosomes (data not shown).

[0083] [Table 4]

[0084] Inhibition of soluble and membrane TNFα-induced cell death: Inhibition of soluble and membrane TNFα-induced cell death by exemplary anti-human TNFα antibodies was evaluated in an in vitro cell-based assay using L929 mouse fibrosarcoma cells that naturally express TNF receptors. When combined with actinomycin-D, TNFα induces classical apoptosis in these cells, resulting in rapid cell death due to the excessive formation of reactive oxygen intermediates that can be rescued by TNFα neutralization. The amount of viable cells can be measured using an MTS-tetrazolium cytotoxicity assay, in which mitochondrial dehydrogenase enzymes in metabolically active cells reduce MTS-tetrazolium to a colored formazan product that can be detected with a microplate reader (Biotek Cytation 5 Imaging Multi-Mode Reader).

[0085] Inhibition of soluble TNFα-induced cell death: To assess the ability of the exemplary anti-human TNFα antibodies to inhibit soluble TNFα-induced cell death, L929 cells were treated separately with either human TNFα or cynomolgus TNFα. L929 cells resuspended at 10,000 cells / 100 μL in assay medium (1×DMEM medium, 10% FBS, 1% Pen-Strep, 1% MEM essential amino acids, 1% L-glutamine, 1% sodium pyruvate) were added to 96-well plates and placed in a tissue culture incubator overnight. The next day, the exemplary antibodies were diluted to concentrations ranging from 15 μg / mL to 0.0005 μg / mL (3-fold dilutions) and 100 μL of each concentration of the exemplary anti-human TNFα antibody was added in duplicate to wells containing one of the following two conditions: 200 pg / mL recombinant human TNFα or 750 pg / mL recombinant cynomolgus monkey TNFα, and the plates were incubated at room temperature for 30 minutes. A human IgG1 isotype control antibody was used as a negative control. The antibody / TNFα / actinomycin-D mixture was then transferred to a 96-well plate containing L929 adherent cells and incubated for 18 hours in a tissue culture incubator. The assay medium was removed and 100 μL of the MTS-tetrazolium substrate mixture was added to the wells and the plates were incubated for 2 hours in a tissue culture incubator. To determine cell death, the plates were read at 490 nm in a microplate reader (Biotek Cytation 5 Imaging Multi-Mode Reader). The results show the concentration at which 50% of TNFα-induced cytotoxicity is inhibited by an exemplary anti-human TNFα antibody (IC 50 , mean ± SEM of two independent experiments) and were calculated using a three-parameter sigmoidal fit of the data (GraphPad Prism9). IC 50 The values ​​are shown in Table 5a.

[0086] Inhibition of membrane TNFα-induced cell death: To evaluate the ability of the exemplary anti-human TNFα antibodies to inhibit membrane TNFα-induced cell death, uncleavable TNFα constructs were stably transfected into Chinese Hamster Ovary (CHO) cells to generate cell surface (membrane) TNFα expressing CHO cells. Uncleavable TNFα constructs were generated with known mutations in the cleavage site of TNFα that allow expression of bioactive TNFα on the cell surface in the absence of TNFα cleavage (Mueller et.al.1999). Incubation of L929 cells with CHO cells expressing uncleavable human TNFα resulted in rapid L929 cell death. To determine whether the exemplary anti-human TNFα antibodies could neutralize the observed cell death, a dose range of 15 μg / mL to 0.0005 μg / mL (3-fold dilutions) was evaluated for each of the exemplary antibodies. Each concentration of exemplary anti-human TNFα antibody (100 μL / well) was added in duplicate to a plate containing 500 CHO TNFα transfectant cells / well + 6.25 μg / mL actinomycin D. The antibody + CHO cell mixture was incubated at room temperature for 30 minutes and then added to the L929 cell plate. A human IgG1 isotype control antibody was used as a negative control and tested at the same dose range as the anti-human TNFα antibodies. L929 cell death was determined essentially as described for the soluble TNFα-induced cell death assay. IC 50 The values ​​are shown in Table 5b.

[0087] The results, as shown in Tables 5a and 5b, demonstrated that the exemplary anti-human TNFα antibodies inhibited soluble human TNFα or soluble cynomolgus monkey TNFα-induced L929 cell killing, and human membrane TNFα-induced L929 cell killing. A dose-dependent inhibition of the cell killing response was observed. Specifically, the IC for inhibition of soluble human TNFα-induced cell killing by the exemplary anti-human TNFα antibodies tested was 0.01. 50(FIG. 5a) ranged from about 0.13 μg / mL to about 0.22 μg / mL, and for inhibition of soluble cyno TNFα-induced cell death ranged from about 0.02 μg / mL to about 0.3 μg / mL. IC for inhibition of human membrane TNFα-induced cell death by exemplary anti-human TNFα antibodies tested 50 (Figure 5b) ranged from about 0.13 µg / mL to about 0.12 µg / mL. The negative control hIgG1 isotype did not inhibit TNFα-induced cell death, as expected.

[0088] [Table 5]

[0089] [Table 6]

[0090] Example 4: Characterization of exemplary anti-human TNFα antibodies that bind to anti-drug antibodies to adalimumab Binding to cynomolgus monkey anti-drug antibodies against adalimumab: Binding of exemplary anti-human TNFα antibodies to anti-drug antibodies against adalimumab (anti-adalimumab antibodies) obtained from affinity purified hyperimmune monkey serum (AP-HIMS) from cynomolgus monkeys hyperimmunized with adalimumab was evaluated. Adalimumab-AffiGel10 was used to purify anti-adalimumab antibodies from adalimumab hyperimmune cynomolgus monkeys. Anti-adalimumab antibodies were detected using titration of AP-HIMS in an ACE-Bridge assay. The assay was developed in accordance with FDA guidance for immunogenicity testing. Briefly, streptavidin-coated 96-well plates (Pierce, 15500) were washed with 1×TBST (Boston BioProducts, IBB-181X) and coated with 30 nM biotinylated adalimumab at 100 μL / well in TBST / 0.1% bovine serum albumin (BSA; Sigma, A7888) for 1 h at room temperature. Plates were washed three times with TBST and affinity-purified anti-adalimumab antibodies were diluted 1:10 in TBS (Fisher, BP2471-1) and added to the coated plates at 100 μL / well and incubated overnight at 4° C. The next day, plates were washed three times with TBST and captured anti-adalimumab antibodies were acid eluted using 65 μL / well of 300 mM acetic acid (Fisher Scientific, A38-500) at room temperature for 5 min. A polypropylene 96-well plate (Corning, 3359) was then loaded with 50 μL of 1 μg / mL each of biotinylated adalimumab and ruthenium-labeled adalimumab in neutralization buffer (0.375 M Tris, 300 mM NaCl, pH 9). 50 μL of the acid elution sample was then added to the polypropylene plate containing the mixture and ADA in neutralization buffer and allowed to crosslink to the labeled antibody for 1 hour at room temperature. MSD Gold 96-well streptavidin plate (Mesoscale, L15SA-1) was washed and blocked with TBS+1% BSA for 1 hour at room temperature, then washed and 80 μL of the crosslinking sample was added to the plate for 1 hour.The plate was washed three times with TBST and 150 μL / well of 2× MSD buffer (Mesoscale, R92TC-2) was added to the plate. The plate was read on an MSD SQ120 reader to obtain the Tier 1 signal expressed as electrochemiluminescent units (ECLU).

[0091] The same AP-HIMS were also tested in an ACE-Bridge assay to detect antibodies against the exemplary anti-human TNFα antibody Ab6, following essentially the same methodology as outlined above for adalimumab, but using biotin and ruthenium-labeled Ab6. The resulting ECLU signal was then plotted as a function of the concentration of AP-HIMS tested.

[0092] The results shown in Figure 1 show that the exemplary anti-human TNFα antibody Ab6 showed low or no binding to anti-drug antibodies against adalimumab purified from serum from cynomolgus monkeys hyperimmunized with adalimumab (maximum ECLU signal 4000) when compared to the binding of adalimumab to its own anti-drug antibodies (maximum ECLU signal 40000). Specifically, the results showed that the exemplary anti-human TNFα antibody Ab6 recognized only about 10% of the anti-drug antibodies against adalimumab generated by cynomolgus monkeys, suggesting that this binding is likely due to shared sequences located away from the CDR regions, such as the antibody constant regions.

[0093] Binding to human patient anti-drug antibodies against adalimumab: Binding of exemplary anti-human TNFα antibodies to anti-drug antibodies against adalimumab (anti-adalimumab antibodies) in 21 patient serum samples obtained from adalimumab-treated patients enrolled in study RA-BEAM was evaluated. The 21 serum samples were collected after baseline and confirmed to have high ADA titers against adalimumab by using methods essentially as described for cynomolgus monkey ADA evaluation. The 21 serum samples were then evaluated for binding to exemplary anti-human TNFα antibody Ab6 using methods essentially as described for cynomolgus monkey ADA evaluation.

[0094] The results shown in Figure 2 indicate that the exemplary anti-human TNFα antibody Ab6 showed low or no binding to anti-drug antibodies to adalimumab in 16 of 21 patient samples tested (ECLU signals were below the assay cutoff point (102 ECLU)). In determining immunogenicity, the cutoff point is the threshold used to identify "presumptive positive" or anti-drug antibody-containing samples. As shown in Figure 2, 5 of the 21 samples had ECLU signals above the cutoff point, but all were less than 20% above the cutoff point and therefore were determined to be within the variability of the assay. The significantly lower or absent binding of anti-human TNFα antibody Ab6 to anti-drug antibodies against adalimumab in human patients treated with adalimumab indicated that the Ab6 and adalimumab antibody sequences were sufficiently different such that ADAs generated against adalimumab by the tested human subjects that are specific for epitopes present in adalimumab are not shared by Ab6 and are therefore poorly recognized by Ab6. These results indicate the potential use of the exemplary anti-human TNFα antibodies for the treatment of patients experiencing reduced clinical responses or adverse reactions to anti-drug antibodies against other TNFα therapeutics such as adalimumab.

[0095] Example 5: Immunogenicity evaluation DC internalization assays, MAPPS assays, and T cell proliferation assays against LCDR1 and HCDR3 peptide clusters were performed to assess the immunogenicity risk of the exemplary TNFα antibodies.

[0096] Dendritic cell internalization assay: The ability of human CD14+ monocytes derived from dendritic cells (DCs) to internalize an exemplary anti-human TNFα antibody was assessed. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs), cultured, and differentiated into immature dendritic cells (with IL-4 and GM-CSF), all using standard protocols. To obtain mature DCs, cells were treated with 1 μg / mL LPS for 4 hours.

[0097] An exemplary anti-human TNFα antibody was diluted to 8 μg / mL in complete RPMI medium and mixed in equal volumes with the detection probe Fab-TAMRA-QSY7 diluted to 5.33 μg / mL in complete RPMI medium, further incubated for 30 min at 4° C. in the dark for complex formation, then added to immature and mature DC cultures and incubated for 24 h at 37° C. in a CO2 incubator. Cells were washed with 2% FBS PBS and resuspended in 100 μL of 2% FBS PBS containing Cytox Green live / dead dye. Data were collected on a BD LSR Fortessa X-20 and analyzed with FlowJo. Live single cells were gated and the percentage of TAMRA fluorescence positive cells was recorded as readout. A normalized internalization index was used to allow comparison of molecules with data generated from different donors. The internalization signal was normalized to the IgG1 isotype (normalized internalization index=0) and the internal positive control PC (normalized internalization index=100) using equation 1:

[0098]

number

[0099] The results, shown in Table 6, indicate that the anti-human TNFα antibodies tested were internalized within the cells upon binding to TNFα on immature and mature dendritic cells.

[0100] [Table 7]

[0101] MHC-Associated Peptide Proteomics (MAPP) Assay: MAPP profiles MHC-II presented peptides on human dendritic cells pretreated with an exemplary anti-human TNFα antibody. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs), cultured, and differentiated into immature dendritic cells (with IL-4 and GM-CSF) using standard protocols. Exemplary antibodies were added to the immature dendritic cells on day 4, and fresh medium containing LPS was replaced after 5 hours of incubation to transform the cells into mature dendritic cells. The next day, mature dendritic cells were lysed in RIPA with protease inhibitors and DNAse. Immunoprecipitation of MHC-II complexes was performed using biotinylated anti-MHC-II antibodies bound to streptavidin beads. Bound complexes were eluted and filtered. Isolated MHC-II peptides were analyzed by mass spectrometry. Peptide identifications were generated by an internal proteomics pipeline using a search algorithm with no enzyme search parameters against a bovine / human database with test sequences added to the database. Peptides identified from the exemplary antibodies were aligned against the parent sequence.

[0102] The results, shown in Table 7, showed that the exemplary anti-human TNFα antibodies had varying degrees of presentation by MAPPs. Ab1 demonstrated the lowest MAPP presentation with one non-germline cluster in three of the ten donors tested.

[0103] [Table 8]

[0104] T cell proliferation assay: The ability of the exemplary anti-human TNFα antibody MAPP-derived peptide cluster to activate CD4+ T cells by inducing cell proliferation was evaluated. CD8+ T cells were depleted from cryopreserved PBMCs from 10 healthy donors and labeled with 1 μM Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE). CD8+ T cell depleted PBMCs were cultured at 4×10 in AIM-V medium (Life Technologies, Catalog No. 12055-083) containing 5% CTS™ Immune Cell SR (Gibco, Catalog No. A2596101). 6 Cells were seeded at 1000 cells / mL / well and tested in triplicate in 2.0 mL containing the following different molecules: DMSO control, medium control, keyhole limpet hemocyanin (KLH; positive control), PADRE-X peptide (synthetic vaccine helper peptide, positive peptide control), or each anti-human TNFα antibody MAPP-derived peptide cluster (10 μM of each peptide). Cells were cultured and incubated for 7 days at 37° C. with 5% CO2. On day 7, samples were stained with the following cell surface markers for viability detection by flow cytometry using a BD LSRFortessa™ equipped with a High Throughput Sampler (HTS): anti-CD3, anti-CD4, anti-CD14, anti-CD19, and DAPI. Data were analyzed using FlowJo® software (FlowJo, LLC, TreeStar) and the Cellular Division Index (CDI) was calculated. Briefly, the CDI for each MAPP-derived peptide cluster was calculated based on the number of proliferating CFSE cells from peptide-stimulated wells. dim The percentage of CD4+ T cells was determined by multiplying CFSE dimThe donor frequency was calculated by dividing by the percentage of CD4+ T cells. A CDI of 2.5 or greater was considered to represent a positive response. Donor frequency percentages across all donors were assessed.

[0105] The results shown in Tables 8a and 8b indicate that the LCDR1 (Table 8a) and HCDR3 (Table 8b) peptides for Ab2 induced T cell response frequencies in approximately 22.0% and 25% of donors, respectively, indicating a significantly reduced immunogenicity risk for Ab2 when compared to the positive controls. The KLH positive control induced T cell responses in 100% of donors, and the PADRE-X (synthetic vaccine helper peptide) positive control induced T cell responses in 67% and 62.5% of donors in the two studies, respectively. This is within the expected range of this assay (positive donor frequency of 48.1% + 24.4).

[0106] [Table 9]

[0107] [Table 10]

[0108] Example 6. Biophysical Properties of Exemplary Anti-Human TNFα Antibodies The biophysical properties of exemplary anti-human TNFα antibodies were evaluated for developability.

[0109] Viscosity: Exemplary anti-human TNFα antibody samples were concentrated to approximately 125 mg / mL in a common formulation buffer matrix at pH 6. The viscosity of each antibody was measured using a VROC® initium (RheoSense) at 15° C. using an average of nine replicate measurements. As shown in Table 9, the results showed that exemplary anti-human TNFα antibodies Ab1 (9.7 cP), Ab2 (9.2 cP), Ab3 (11.4 cP), and Ab4 (10.6 cP) had good viscosity profiles for developability.

[0110] Thermal stability: Differential scanning calorimetry (DSC) was used to evaluate the stability of the exemplary antibodies against thermal denaturation. The thermal melting temperatures of the antibodies in PBS, pH 7.2 buffer obtained by data fitting when unresolved are listed in Table 9 (Tonset, TM1, TM2, and TM3). Although the thermal transitions for each domain were not all fully resolved, the data shown in Table 9 indicates that the exemplary anti-human TNFα antibodies Ab1, Ab2, Ab3, and Ab4 had good thermal stability profiles for development.

[0111] Aggregation due to temperature stress: Solution stability over time of exemplary antibodies was evaluated at approximately 100 mg / mL. Samples were incubated at 5° C. and 35° C. for a period of 28 weeks. After incubation, samples were analyzed for percent high molecular weight (% HMW) species using size exclusion chromatography (SEC). The results, shown in Table 9, indicate that exemplary anti-human TNFα antibodies Ab1, Ab2, Ab3, and Ab4 had good aggregation profiles for developability.

[0112] [Table 11]

[0113] Example 7: In vivo characterization of anti-human TNFα antibodies Inhibition of human TNFα-induced CXCL1 cytokine production in vivo: Neutralization of TNFα-induced CXCL1 by exemplary anti-human TNFα antibodies was evaluated in vivo. Administration of human TNFα to C57 / B6 mice induces a rapid and transient increase in mouse plasma CXCL1 levels. This allows the investigation of the neutralizing capacity of exemplary anti-human TNFα antibodies in vivo.

[0114] Briefly, C57 / B6 mice (N=8 / group) were subcutaneously dosed with 0.3 mg / kg or 3 mg / kg of an exemplary antibody or 3 mg / kg of a non-binding isotype control. 24 hours after antibody administration, mice were challenged by intraperitoneal injection of human TNFα at a dose of 3 μg / mouse. Two hours after human TNFα challenge, mice were sacrificed and blood was collected and clarified to plasma by centrifugation. Plasma was analyzed for mouse CXCL1 concentration using a commercially available MSD assay (MesoScale Discovery, P / N. number K152QTG-1) according to the manufacturer's instructions.

[0115] The results shown in Table 10 indicate that the exemplary anti-human TNFα antibodies significantly inhibited human TNFα-induced plasma CXCL1 production in vivo in a dose-dependent manner compared to isotype control-treated mice (p<0.05, ANOVA followed by Turkey's multiple comparison test). In particular, the exemplary anti-human TNFα antibodies inhibited TNFα-induced in vivo plasma CXCL1 production by about 82% to about 93% at 3 mg / kg and about 46.5% to about 64.5% at 0.3 mg / kg. Thus, demonstrating that the exemplary anti-human TNFα antibodies neutralized the biological effects induced by human TNFα in vivo.

[0116] [Table 12]

[0117] Sequence Listing Ab1 SEQ ID NO: 1 HCDR1 for Ab1, Ab2, and Ab6 GYTFTGYYIH SEQ ID NO: 2 HCDR2 for Ab1, Ab2, and Ab6 WINPYTGGTNYAQKFQG SEQ ID NO: 3 HCDR3 for Ab1 DLYGSSNYGGDV SEQ ID NO: 4 LCDR1 for Ab1 and Ab3 QASQGISNYLN SEQ ID NO: 5 LCDR2 for Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET SEQ ID NO: 6 LCDR3 for Ab1, Ab3, and Ab5 QQYDKLPLT SEQ ID NO: 7 VH for Ab1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGGDVWGQGTTVTVSS SEQ ID NO: 8 VL for Ab1 and Ab3 DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGGTKVEIK SEQ ID NO: 9 HC for Ab1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGGDVWG QGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10 LC for Ab1 and Ab3 DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:11 HC DNA for Ab1 SEQ ID NO: 12 LC DNA for Ab1 and Ab3 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAAGCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Ab2 SEQ ID NO: 1 HCDR1 for Ab1, Ab2, and Ab6 GYTFTGYYIH SEQ ID NO: 2 HCDR2 for Ab1, Ab2, and Ab6 WINPYTGGTNYAQKFQG SEQ ID NO: 13 HCDR3 for Ab2, Ab3, Ab4, and Ab5 DLYGSSNYGMDV SEQ ID NO: 14 LCDR1 for Ab2, Ab4, and Ab5 QASQGIRNYLN SEQ ID NO: 5 LCDR2 for Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET SEQ ID NO: 15 LCDR3 for Ab2 and Ab4 QQYDNLPLT SEQ ID NO: 16 VH for Ab2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSS SEQ ID NO: 17 VL for Ab2 and Ab4 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK SEQ ID NO:18 HC for Ab2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWG QGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 19 LC for Ab2 and Ab4 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:20 HC DNA for Ab2 SEQ ID NO:21 LC DNA for Ab2 and Ab4 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTCGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAACCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Ab3 SEQ ID NO: 22 HCDR1 for Ab3, Ab4, and Ab5 GYTFTGYYMH SEQ ID NO: 23 HCDR2 for Ab3, Ab4, and Ab5 WINPYTGGTKYAQKFQG SEQ ID NO: 13 HCDR3 for Ab2, Ab3, Ab4, and Ab5 DLYGSSNYGMDV SEQ ID NO: 4 LCDR1 for Ab2, Ab4, and Ab5 QASQGISNYLN SEQ ID NO: 5 LCDR2 for Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET SEQ ID NO: 6 LCDR3 for Ab1, Ab3, and Ab5 QQYDKLPLT SEQ ID NO: 24 VH for Ab3, Ab4 and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSS SEQ ID NO: 8 VL for Ab1 and Ab3 DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGGTKVEIK SEQ ID NO: 25 HC for Ab3, Ab4 and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWG QGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10 LC for Ab1 and Ab3 DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:26 HC DNA for Ab3, Ab4, and Ab5 SEQ ID NO: 12 LC DNA for Ab1 and Ab3 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAAGCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Ab4 SEQ ID NO: 22 HCDR1 for Ab3, Ab4, and Ab5 GYTFTGYYMH SEQ ID NO: 23 HCDR2 for Ab3, Ab4, and Ab5 WINPYTGGTKYAQKFQG SEQ ID NO: 13 HCDR3 for Ab2, Ab3, Ab4, and Ab5 DLYGSSNYGMDV SEQ ID NO: 14 LCDR1 for Ab2, Ab4, and Ab5 QASQGIRNYLN SEQ ID NO: 5 LCDR2 for Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET SEQ ID NO: 15 LCDR3 for Ab2 and Ab4 QQYDNLPLT SEQ ID NO: 24 VH for Ab3, Ab4 and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSS SEQ ID NO: 17 VL for Ab2 and Ab4 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK SEQ ID NO: 25 HC for Ab3, Ab4 and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWG QGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 19 LC for Ab2 and Ab4 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:26 HC DNA for Ab3, Ab4, and Ab5 SEQ ID NO:21 LC DNA for Ab2 and Ab4 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTCGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAACCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Ab5 SEQ ID NO: 22 HCDR1 for Ab3, Ab4, and Ab5 GYTFTGYYMH SEQ ID NO: 23 HCDR2 for Ab3, Ab4, and Ab5 WINPYTGGTKYAQKFQG SEQ ID NO: 13 HCDR3 for Ab2, Ab3, Ab4, and Ab5 DLYGSSNYGMDV SEQ ID NO: 14 LCDR1 for Ab2, Ab4, and Ab5 QASQGIRNYLN SEQ ID NO: 5 LCDR2 for Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET SEQ ID NO: 6 LCDR3 for Ab1, Ab3, and Ab5 QQYDKLPLT SEQ ID NO: 24 VH for Ab3, Ab4 and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSS SEQ ID NO: 27 VL for Ab5 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGGTKVEIK SEQ ID NO: 25 HC for Ab3, Ab4 and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWG QGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:28 LC for Ab5 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:26 HC DNA for Ab3, Ab4, and Ab5 SEQ ID NO:29 LC DNA for Ab5 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTCGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAAGCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Ab6 SEQ ID NO: 1 HCDR1 for Ab1, Ab2, and Ab6 GYTFTGYYIH SEQ ID NO: 2 HCDR2 for Ab1, Ab2, and Ab6 WINPYTGGTNYAQKFQG SEQ ID NO: 30 HCDR3 for Ab6 DIYGSSNYGGDV SEQ ID NO: 31 LCDR1 for Ab6 QASQDISNYLN SEQ ID NO: 5 LCDR2 for Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET SEQ ID NO: 32 LCDR3 for Ab6 QQYDTLPLT SEQ ID NO: 33 VH for Ab6 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDIYGSSNYGGDVWGQGTTVTVSS SEQ ID NO: 34 VL for Ab6 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDTLPLTFGGGTKVEIK SEQ ID NO: 35 HC for Ab6 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDIYGSSNYGGDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 36 LC for Ab6 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDTLPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:37 HC DNA for Ab6 SEQ ID NO:38 LC DNA for Ab6 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATACCCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC SEQ ID NO: 39 Human TNFα protein MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANG VELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO:40 Rhesus TNFα protein MSTESMIRDVELAEEALPRKTAGPQGSRRCWFLSLFSFLLVAGATTLFCLLHFGVIGPQREEFPKDPSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANG VELTDNQLVVPSEGLYLIYSQVLFKGQGCPSNHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINLPDYLDFAESGQVYFGIIAL SEQ ID NO: 41 Mouse TNFα protein MSTESMIRDVELAEEALPQKMGGFQNSRRCLCLSLFSFLLVAGATTLFCLLLNFGVIGPQRDEKFPNGLPLISSMAQTLTLRSSSQNSSDKPVAHVVANHQVEEQLEWLSQRANALLA NGMDLKDNQLVVPADGLYLVYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVNLLSAVKSPCPKDTPEGAELKPWYEPIYLGGVFQLEKGDQLSAEVNLPKYLDFAESGQVYFGVIAL SEQ ID NO: 42 Rat TNFα protein MSTESMIRDVELAEEALPKKMGGLQNSRRCLCLSLFSFLLVAGATTLFCLLNFGVIGPNKEEKFPNGLISSMAQTLTLRSSSQNSSDKPVAHVVANHQAEEQLEWLSQRANALLA NGMDLKDNQLVVPADGLYLIYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVSLLSAIKSPCPKDTPEGAELKPWYEPMYLGGVFQLEKGDLLSAEVNLPKYLDITESGQVYFGVIAL SEQ ID NO: 43 Rabbit TNFα protein MSTESMIRDVELAEGPLPKKAGGPQGSKRCLCLSLFSFLLVAGATTLFCLLHFRVIGPQEEESPNNLHLVNPVAQMVTLRSASRALSDKPLAHVVANPQVEGQLQWLSQRANALLAN GMKLTDNQLVVPADGLYLIYSQVLFSGQGCRSYVLLTHTVSRFAVSYPNKVNLLSAIKSPCHRETPEEAEPMAWYEPIYLGGVFQLEKGDRLSTEVNQPEYLDLAESGQVYFGIIAL SEQ ID NO: 44 Canine TNFα protein MSTESMIRDVELAEEPLPKKAGGPPGSRRCFCLSLFSFLLVAGATTLFCLLHFGVIGPQREELPNGLQLISPLAQTVKSSSRTPSDKPVAHVVANPEAEGQLQWLSRRANALLANG VELTDNQLIVPSDGLYLIYSQVLFKGQGCPSTHVLLTHTISRFAVSYQTKVNLLSAIKSPCQRETPEGTEAKPWYEPIYLGGVFQLEKGDRLSAEINLPNYLDFAESGQVYFGIIAL SEQ ID NO: 45 Cynomolgus TNFα protein MSTESMIQDVELAEEALPRKTAGPQGSRRCWFLSLFSFLLVAGAATLFCLLHFGVIGPQREEFPKDPSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALVANG VELTDNQLVVPSEGLYLIYSQVLFKGQGCPSNHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINLPDYLDFAESGQVYFGIIAL SEQ ID NO: 46 LCDR1 consensus sequence QASQGIXaa7NYLN wherein Xaa7 is serine or arginine SEQ ID NO: 47 LCDR3 consensus sequence QQYDXaa5LPLT wherein Xaa5 is asparagine or lysine SEQ ID NO: 48 Human TNFR1 MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPPGGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVE ISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSL LFIGLMYRYQRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTLGFSPVPSSTFTSSSTYTPGDCPNFAAPRREVAPPYQGADPILATALASDPIPNPLQKW EDSAHKPQSLTDTDDPATLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYSMLATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR SEQ ID NO: 49 Human TNFR2 MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQN RICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPT PEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTR NQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS

Claims

1. An antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; said HCDR1 comprising SEQ ID NO:1; said HCDR2 comprising SEQ ID NO:2; said HCDR3 comprising SEQ ID NO:3; said LCDR1 comprising SEQ ID NO:4; the LCDR2 comprises SEQ ID NO:5; An antibody wherein the LCDR3 comprises SEQ ID NO:

6.

2. The antibody of claim 1, wherein the VH comprises SEQ ID NO:7 and the VL comprises SEQ ID NO:

8.

3. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprising SEQ ID NO: 9, and the LC comprising SEQ ID NO:

10.

4. An antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; said HCDR1 comprising SEQ ID NO:1; said HCDR2 comprising SEQ ID NO:2; the HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprising SEQ ID NO: 14; the LCDR2 comprises SEQ ID NO:5; An antibody wherein the LCDR3 comprises SEQ ID NO:

15.

5. The antibody of claim 4, wherein the VH comprises SEQ ID NO: 16 and the VL comprises SEQ ID NO:

17.

6. The antibody of claim 4 or 5, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprising SEQ ID NO: 18, and the LC comprising SEQ ID NO:

19.

7. An antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; said HCDR1 comprising SEQ ID NO:22; said HCDR2 comprising SEQ ID NO:23; the HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprising SEQ ID NO:4; the LCDR2 comprises SEQ ID NO:5; An antibody wherein the LCDR3 comprises SEQ ID NO:

6.

8. An antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; said HCDR1 comprising SEQ ID NO:22; said HCDR2 comprising SEQ ID NO:23; the HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprising SEQ ID NO: 14; the LCDR2 comprises SEQ ID NO:5; An antibody wherein the LCDR3 comprises SEQ ID NO:

15.

9. An antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; said HCDR1 comprising SEQ ID NO:22; said HCDR2 comprising SEQ ID NO:23; the HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprising SEQ ID NO: 14; the LCDR2 comprises SEQ ID NO:5; An antibody wherein the LCDR3 comprises SEQ ID NO:

6.

10. An antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; said HCDR1 comprising SEQ ID NO:22; said HCDR2 comprising SEQ ID NO:23; the HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprising SEQ ID NO:46; the LCDR2 comprises SEQ ID NO:5; An antibody wherein the LCDR3 comprises SEQ ID NO:

6.

11. An antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; said HCDR1 comprising SEQ ID NO:22; said HCDR2 comprising SEQ ID NO:23; the HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprising SEQ ID NO: 14; the LCDR2 comprises SEQ ID NO:5; An antibody wherein the LCDR3 comprises SEQ ID NO:

47.

12. The antibody of any one of claims 7 to 11, wherein the VH comprises SEQ ID NO: 24 and the VL comprises SEQ ID NO: 8, 17, or 27.

13. The antibody of any one of claims 7 to 12, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprising SEQ ID NO: 25, and the LC comprising SEQ ID NO: 10, 19, or 28.

14. An antibody that binds to human TNFα, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; said HCDR1 comprising SEQ ID NO:1; said HCDR2 comprising SEQ ID NO:2; the HCDR3 comprises SEQ ID NO: 30; said LCDR1 comprising SEQ ID NO:31; the LCDR2 comprises SEQ ID NO:5; An antibody wherein the LCDR3 comprises SEQ ID NO:

32.

15. The antibody of claim 14, wherein the VH comprises SEQ ID NO: 33 and the VL comprises SEQ ID NO:

34.

16. The antibody of claim 14 or 15, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprising SEQ ID NO: 35, and the LC comprising SEQ ID NO:

36.

17. An antibody comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 9, 18, 25, or 35, and the LC comprises SEQ ID NO: 10, 19, 28, or 36.

18. The antibody comprises a light chain and a heavy chain, the heavy chain comprising: A cysteine ​​at amino acid residue 124 (EU numbering), a cysteine ​​at amino acid residue 378 (EU numbering), or 16. The antibody of any one of claims 1 or 2, 4 or 5, 7 to 12, 14 or 15, comprising a cysteine ​​at amino acid residue 124 (EU numbering) and a cysteine ​​at amino acid residue 378 (EU numbering).

19. The antibody of any one of claims 1 or 2, 4 or 5, 7 to 12, 14 or 15, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), and the HC is of human IgG1 isotype.

20. A nucleic acid comprising a sequence encoding SEQ ID NO: 9, 10, 18, 19, 25, 28, 35, or 36.

21. A vector comprising the nucleic acid of claim 20.

22. 22. The vector of claim 21, wherein the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 9, 18, 25, or 35 and a second nucleic acid sequence encoding SEQ ID NO: 10, 19, 28, or 36.

23. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:9, 18, 25, or 35, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:10, 19, 28, or 36.

24. A cell comprising the vector of claim 21 or 22.

25. The cell of claim 24 , wherein the cell is a mammalian cell.

26. 26. A method for producing an antibody, comprising culturing a cell according to claim 24 or 25 under conditions in which the antibody is expressed, and then recovering the expressed antibody from the culture medium.

27. 27. An antibody produced by the method of claim 26.

28. 28. An antibody drug conjugate comprising the antibody of any one of claims 1 to 19 or 27.

29. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 19 or 27 and a pharma- ceutically acceptable excipient, diluent or carrier.

30. 30. A method of treating a TNFα-related disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody of any one of claims 1 to 19 or 27, or a pharmaceutical composition of claim 29.

31. 31. The method of claim 30, wherein the TNFα-related disorder is a chronic autoinflammatory immune disorder.

32. 32. The method of claim 31, wherein the chronic autoinflammatory immune disorder is selected from rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, or Behcet's disease.

33. The method of claim 30, wherein the subject receiving the therapeutically effective amount of the antibody has been previously treated with another anti-TNFα therapeutic, and the subject has generated anti-drug antibodies against the other anti-TNFα therapeutic.

34. 34. The method of claim 33, wherein the other anti-TNFα therapeutic agent is selected from adalimumab, infliximab, golimumab, certolizumab, or etanercept.

35. 34. The method of claim 33, wherein the antibody has low or no binding to anti-drug antibodies against adalimumab.

36. The antibody of any one of claims 1 to 19 or 27 for use in therapy.

37. The antibody of any one of claims 1 to 19 or 27, or the pharmaceutical composition of claim 29, for use in the treatment of a TNFα-related disorder.

38. 38. The antibody or pharmaceutical composition for use according to claim 37, wherein the TNFα-related disorder is a chronic autoinflammatory immune disorder.

39. 39. The antibody or pharmaceutical composition for use according to claim 38, wherein the chronic autoinflammatory immune disorder is selected from rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, or Behcet's disease.

40. 30. Use of an antibody according to any one of claims 1 to 19 or 27 in the manufacture of a medicament for the treatment of a TNFα-related disorder.

41. 41. The use of claim 40, wherein the TNFα-related disorder is a chronic autoinflammatory immune disorder.

42. 42. The use of claim 41, wherein the chronic autoinflammatory disorder is selected from rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, or Behcet's disease.

43. The antibody of any one of claims 1 to 19 or 27, wherein the antibody neutralizes human TNFα.

44. The antibody of any one of claims 1 to 19 or 27, wherein the antibody is an internalizing antibody.

45. The antibody of any one of claims 1 to 19 or 27, wherein the antibody has low immunogenicity.

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