Anti-TNFα antibodies and compositions

Abstract

The present invention relates to anti-TNFα antibodies and methods of their use in treating diseases and conditions associated with TNFα activity, such as autoimmune or inflammatory diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 356,138, filed June 28, 2022. The disclosure of this priority application is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, the entire contents of which are incorporated herein by reference. The electronic copy of the Sequence Listing, created on June 21, 2023, is designated 123314.WO003.xml and is 170,275 bytes in size. [Background technology]

[0003] Background of the Invention TNFα is a pleiotropic, proinflammatory cytokine expressed by cells of the immune system, including monocytes / macrophages (de Waal Malefyt et al., J Exp Med. (1991) 174:1209-20), dendritic cells (DCs) (Ho et al., J Immunol. (2001) 166:1499-506), lymphocytes (Brehm et al., J. Immunol. (2005) 175: 5043-49; Fauriat et al., Blood (2010) 115: 2167-76; Williamson et al., Proc Natl Acad Sci. USA (1983) 80:5397-401), and neutrophils (Coulthard et al., Clin Exp Immunol. (2012) 170:36-46). It is synthesized on the cell membrane as a transmembrane protein and subsequently proteolyzed by TNFα-converting enzyme (TACE) to release the soluble TNFα homotrimeric protein (Sedger and McDermott, Cytokine & Growth Factor Reviews (2014) 25:453-72). TNFα is a potent mediator of inflammation and is involved in the development of inflammatory and autoimmune diseases (Kalliolias and Ivashkiv, Nat Rev Rheumatol. (2016) 12:49-62).

[0004] TNFα is a well-documented therapeutic target, and several TNFα antibodies (infliximab, adalimumab, golimumab, and certolizumab) have been approved for the treatment of certain rheumatoid and inflammatory bowel diseases (IBD). Although these antibodies have dramatically improved outcomes in rheumatoid diseases, all four antibodies have significant immunogenicity (van Schouenburg et al., Nat Rev Rheumatol. (2013) 9:164-72). Immunogenicity is associated with reduced drug concentrations, which in turn is associated with treatment discontinuation, reduced efficacy, or treatment failure (Adedokun et al., J. Crohn's Colitis (2017) 11:35-46; Adedokun et al., Inflamm Bowel Dis. (2019) 25:1532-40; Atiqi et al., Frontiers Immunol. (2020) 11:312; Bartelds et al., JAMA (2011) 305:1460-8; Gorovits et al., Clinical & Experimental Immunology (2018) 192:348-65; Jani et al., Ann Rheum Dis (2017) 76:208-13; Kennedy et al., Lancet Gastroenterol. Hepatol. (2019) 4:341-53; Radstake et al., Ann Rheum Dis (2009) 68:1739-45; van Schouenburg et al., supra). Optimization of treatment regimens (e.g., dosage, frequency of administration, concomitant use of immunomodulatory agents, etc.) reduces, but does not resolve, the immunogenicity problem (Atiqi et al., supra).

[0005] The exact molecular mechanism of immunogenicity of TNFα antibodies is unclear, and antibodies directed against TNFα are inherently more likely to elicit a greater immune response than antibodies directed against other targets. The FDA-approved TNFα antibodies infliximab (chimeric antibody), certolizumab (humanized antibody), adalimumab (human antibody), and golimumab (human antibody) all exhibit significant immunogenicity, despite varying degrees of homology of their protein sequences with human antibodies. In contrast, two non-TNFα therapeutic antibodies approved for the treatment of certain rheumatic and inflammatory bowel diseases, vedolizumab (anti-α4β7) and ustekinumab (anti-IL-12 / 23), bind to membrane-associated and soluble targets, respectively, and do not elicit significant immunogenicity (Hanauer et al., J Crohn's Colitis (2019) 14:23-32; Sandborn et al., Gastroenterology (2019) 156: Supplement 1, S-1097, AGA Abstract Tu1718; Van den Berghe et al., J Gastro Hepatol. (2018) 34:1175-81; Wyant et al., J Clin Pharmacol. (2021) 61:1174-81).

[0006] Two characteristics of the target protein, TNFα, may contribute to the immunogenicity of the entire class of anti-TNFα antibodies. First, because TNFα is expressed as a homotrimeric protein, soluble TNFα can form immune complexes (ICs) of various sizes with antibodies depending on their relative stoichiometry. Large ICs are multivalent lattices with different antigen-antibody ratios that bind IgG receptors with high affinity and are internalized into processing pathways that promote MHC class I cross-presentation and MHC class II-restricted epitope presentation (Baker et al., Cell Mol Life Sci (2013) 70: 1319-34; Krishna and Nadler, Front Immunol. (2016) 7:21; Weflen et al., Mol Biol Cell (2013) 24:2398-405). As a result, large ICs are potent drivers of immunogenicity, and preformation of ICs has long been used as a strategy to enhance immune responses (Terres and Wolins, J Immunol. (1961) 86:361-8; Morrison and Terres, J Immunol. (1966) 96:901-5; Klaus, Immunology (1978) 34: 643-52). More recently, ICs have been shown to play an important role in immunity to anti-TNFα antibodies in mice (Arnoult et al., J Immunol. (2017) 199:418-24).

[0007] A second feature of TNFα that may contribute to the enhanced immunogenicity of anti-TNFα antibodies is their expression on the cell membrane of antigen-presenting cells of the immune system, including dendritic cells (DCs). Membrane-bound TNFα (mTNFα) may enable internalization and delivery of TNFα antibodies to endocytic compartments. Antibody-based targeting of membrane proteins on DCs has been utilized as a strategy to induce rapid immune responses (Chen et al., Human Vaccines Immunotherapeutics (2016) 12:612-22; Wang et al., Proc Natl Acad Sci. USA (2000) 96:847-52). In this context, it has recently been demonstrated that antibodies bound to mTNFα expressed on dendritic cells are rapidly internalized into endosomes, transported to lysosomes, and digested, with the antibody peptides then presented by MHC class II molecules (Deora et al., MABS (2017) 9:680-95). Furthermore, tetanus toxin peptides fused to anti-TNFα antibodies were also presented by DCs to initiate T cell recall proliferative responses (Deora et al., supra).

[0008] A less immunogenic TNFα antibody may maintain more stable serum antibody levels, leading to fewer treatment failures and less need for treatment discontinuation or switching to alternative therapeutic agents.

[0009] Given the important role of TNFα in the pathogenesis of autoimmune and inflammatory diseases, new and improved immunotherapies that target TNFα are needed. Summary of the Invention

[0010] Summary of the Invention The present invention is directed to novel anti-TNFα antibodies, pharmaceutical compositions comprising one or more of these antibodies, and the use of the antibodies and pharmaceutical compositions for the treatment of autoimmune and inflammatory diseases. In some embodiments, the antibodies of the present invention are variants of well-characterized, clinically validated anti-TNFα antibodies engineered to enhance dissociation from TNFα at acidic pH and prevent the formation of large ICs. These characteristics are expected to reduce transport to lysosomes after binding to soluble or membrane-bound TNFα, thereby reducing immunogenicity. Compared to currently available therapies for autoimmune and inflammatory diseases, including antibody therapy, it is contemplated that the antibodies of the present invention may provide superior clinical responses, either alone or in combination with other therapies, to treat autoimmune and / or inflammatory diseases.

[0011] In some aspects, the present invention provides a method for producing a pharmaceutical composition comprising: a) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8; b) a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 12; or c) VH comprising the amino acid sequence of SEQ ID NO: 14 and VL comprising the amino acid sequence of SEQ ID NO: 16 providing an anti-TNFα antibody or antigen-binding portion thereof that binds to the same epitope of human TNFα as a reference antibody comprising wherein the anti-TNFα antibody or antigen-binding portion comprises a VH and VL that are at least 90% identical to the VH and VL, respectively, of a reference antibody; and the anti-TNFα antibody or antigen-binding portion has a lower binding affinity for TNFα at pH 6.0 than at pH 7.4. In some embodiments, the anti-TNFα antibody or antigen-binding portion may be monovalent.

[0012] In some embodiments, the anti-TNFα antibody a) a monovalent antigen-binding protein comprising a heavy chain (HC) comprising a VH that is at least 90% identical to the VH of a reference antibody and a light chain (LC) comprising a VL that is at least 90% identical to the VL of the reference antibody; and b) a truncated HC lacking the variable and CH1 domains Including, wherein the antigen-binding protein HC and the truncated HC can dimerize. In certain embodiments, the antigen-binding protein HC and the truncated HC may comprise knob-in-hole modifications, for example, the antigen-binding protein HC is of isotype subclass IgG1 and comprises mutations T366S, L368A, and Y407A in the CH3 domain, and the truncated HC is of isotype subclass IgG1 and comprises mutation T366W in the CH3 domain, with residues numbered according to the Eu system. Additionally or alternatively, the antigen-binding protein HC may be of isotype subclass IgG1 and comprises mutation Y349C, and / or the truncated HC may be of isotype subclass IgG1 and comprises mutation S354C, with residues numbered according to the Eu system.

[0013] The present invention also provides a) SEQ ID NOs: 87, 76, 97, 88, 89 and 90, respectively; b) SEQ ID NOs: 87, 76, 98, 88, 89 and 90, respectively; c) SEQ ID NOs: 87, 76, 101, 88, 89 and 90, respectively; d) SEQ ID NOs: 87, 76, 102, 88, 89 and 103, respectively; e) SEQ ID NOs: 87, 76, 77, 104, 89 and 90, respectively; f) SEQ ID NOs: 87, 76, 77, 88, 89 and 105, respectively; g) SEQ ID NOs: 87, 76, 77, 88, 89 and 106, respectively; h) SEQ ID NOs: 87, 76, 77, 107, 89 and 90, respectively; i) SEQ ID NOs: 87, 76, 77, 104, 89 and 108, respectively; j) SEQ ID NOs: 87, 76, 97, 104, 89 and 90, respectively; k) SEQ ID NOs: 87, 76, 97, 88, 89 and 105, respectively; l) SEQ ID NOs: 87, 76, 97, 88, 8 and 106, respectively; m) SEQ ID NOs: 87, 76, 97, 107, 89 and 90, respectively; n) SEQ ID NOs: 87, 76, 98, 104, 89 and 90, respectively; o) SEQ ID NOs: 87, 76, 98, 88, 89 and 105, respectively; p) SEQ ID NOs: 87, 76, 98, 88, 89 and 106, respectively; q) SEQ ID NOs: 87, 76, 98, 107, 89 and 90, respectively; r) SEQ ID NOs: 87, 76, 101, 104, 89 and 90, respectively; s) SEQ ID NOs: 87, 76, 101, 88, 89 and 105, respectively; t) SEQ ID NOs: 87, 76, 101, 88, 89 and 106, respectively; or u) SEQ ID NOs: 87, 76, 101, 107, 89 and 90, respectively The present invention provides an anti-TNFα antibody, or antigen-binding portion thereof, comprising heavy chain (HC) CDR1-3 and light chain (LC) CDR1-3 comprising:

[0014] In some embodiments, the antibody or antigen-binding portion comprises: a) SEQ ID NOs: 30 and 16, respectively; b) SEQ ID NOs: 32 and 16, respectively; c) SEQ ID NOs: 34 and 16, respectively; d) SEQ ID NOs: 36 and 38, respectively; e) SEQ ID NOs: 14 and 40, respectively; f) SEQ ID NOs: 14 and 42, respectively; g) SEQ ID NOs: 14 and 44, respectively; h) SEQ ID NOs: 14 and 46, respectively; i) SEQ ID NOs: 14 and 48, respectively; j) SEQ ID NOs: 30 and 40, respectively; k) SEQ ID NOs: 30 and 42, respectively; l) SEQ ID NOs: 30 and 44, respectively; m) SEQ ID NOs: 30 and 46, respectively; n) SEQ ID NOs: 32 and 40, respectively; o) SEQ ID NOs: 32 and 42, respectively; p) SEQ ID NOs: 32 and 44, respectively; q) SEQ ID NOs: 32 and 46, respectively; r) SEQ ID NOs: 34 and 40, respectively; s) SEQ ID NOs: 34 and 42, respectively; t) SEQ ID NOs: 34 and 44, respectively; or u) SEQ ID NOs: 34 and 46, respectively and a light chain variable domain (VL) comprising:

[0015] The present invention also provides a) SEQ ID NOs: 81, 76, 109, 83, 79 and 80, respectively; b) SEQ ID NOs: 81, 76, 110, 83, 79 and 80, respectively; c) SEQ ID NOs: 81, 76, 111, 83, 79 and 80, respectively; d) SEQ ID NOs: 81, 76, 82, 112, 79 and 80, respectively; e) SEQ ID NOs: 81, 76, 82, 83, 79 and 99, respectively; f) SEQ ID NOs: 81, 76, 82, 83, 79 and 113, respectively; g) SEQ ID NOs: 81, 76, 82, 83, 79 and 100, respectively; h) SEQ ID NOs: 81, 76, 82, 114, 79 and 80, respectively; i) SEQ ID NOs: 81, 76, 82, 83, 79 and 115, respectively; j) SEQ ID NOs: 81, 76, 82, 112, 79 and 116, respectively; k) SEQ ID NOs: 81, 76, 82, 117, 79 and 80, respectively; l) SEQ ID NOs: 81, 76, 82, 118, 79 and 80, respectively; or m) SEQ ID NOs: 81, 76, 82, 119, 79 and 80, respectively The present invention provides an anti-TNFα antibody, or antigen-binding portion thereof, comprising heavy chain (HC) CDR1-3 and light chain (LC) CDR1-3 comprising:

[0016] In some embodiments, the antibody or antigen-binding portion comprises: a) SEQ ID NOs: 50 and 8, respectively; b) SEQ ID NOs: 52 and 8, respectively; c) SEQ ID NOs: 54 and 8, respectively; d) SEQ ID NOs: 6 and 56, respectively; e) SEQ ID NOs: 6 and 58, respectively; f) SEQ ID NOs: 6 and 60, respectively; g) SEQ ID NOs: 6 and 62, respectively; h) SEQ ID NOs: 6 and 64, respectively; i) SEQ ID NOs: 6 and 66, respectively; j) SEQ ID NOs: 6 and 68, respectively; k) SEQ ID NOs: 6 and 70, respectively; l) SEQ ID NOs: 6 and 72, respectively; or m) SEQ ID NOs: 6 and 74, respectively and VH and VL comprising:

[0017] In some embodiments, the anti-TNFα antibody or antigen-binding portion thereof is monovalent. In particular embodiments, the monovalent antibody comprises: a) a monovalent antigen-binding protein comprising an HC comprising the VH of an antibody described herein and an LC comprising the VL of an antibody described herein; and b) a truncated HC lacking the variable and CH1 domains; wherein said antigen-binding protein HC and truncated HC are capable of dimerizing.

[0018] In certain embodiments, the monovalent antibody may comprise: a) a monovalent antigen-binding protein comprising an HC comprising a VH having the amino acid sequence of SEQ ID NO: 50 and an LC comprising a VL having the amino acid sequence of SEQ ID NO: 8; and b) a truncated HC lacking the variable and CH1 domains; wherein said antigen-binding protein HC and truncated HC are capable of dimerizing.

[0019] In certain embodiments, the monovalent antibody may comprise: a) a monovalent antigen-binding protein comprising an HC comprising a VH having the amino acid sequence of SEQ ID NO: 54 and an LC comprising a VL having the amino acid sequence of SEQ ID NO: 8; and b) a truncated HC lacking the variable and CH1 domains; wherein said antigen-binding protein HC and truncated HC are capable of dimerizing.

[0020] In certain embodiments, the monovalent antibody may comprise: a) a monovalent antigen-binding protein comprising an HC comprising a VH having the amino acid sequence of SEQ ID NO: 6 and an LC comprising a VL having the amino acid sequence of SEQ ID NO: 56; and b) a truncated HC lacking the variable and CH1 domains; wherein said antigen-binding protein HC and truncated HC are capable of dimerizing.

[0021] In certain embodiments, the monovalent antibody may comprise: a) a monovalent antigen-binding protein comprising an HC comprising a VH having the amino acid sequence of SEQ ID NO: 6 and an LC comprising a VL having the amino acid sequence of SEQ ID NO: 64; and b) a truncated HC lacking the variable and CH1 domains; wherein said antigen-binding protein HC and truncated HC are capable of dimerizing.

[0022] In certain embodiments, the monovalent antibody may comprise: a) a monovalent antigen-binding protein comprising an HC comprising a VH having the amino acid sequence of SEQ ID NO: 6 and an LC comprising a VL having the amino acid sequence of SEQ ID NO: 68; and b) a truncated HC lacking the variable and CH1 domains; wherein said antigen-binding protein HC and truncated HC are capable of dimerizing.

[0023] In certain embodiments, the monovalent antibody may comprise: a) a monovalent antigen-binding protein comprising an HC comprising a VH having the amino acid sequence of SEQ ID NO: 14 and an LC comprising a VL having the amino acid sequence of SEQ ID NO: 44; and b) a truncated HC lacking the variable and CH1 domains; wherein said antigen-binding protein HC and truncated HC are capable of dimerizing.

[0024] In some embodiments of the above heterotrimeric monovalent antibodies, the antigen binding protein HC and the truncated HC comprise knobs-into-holes modifications, for example, wherein said antigen binding protein HC is of isotype subclass IgG1 and comprises mutations T366S, L368A, and Y407A in the CH3 domain, and said truncated HC is of isotype subclass IgG1 and comprises mutation T366W in the CH3 domain, and residues are numbered according to the Eu system. Additionally or alternatively, the antigen binding protein HC may be of isotype subclass IgG1 and comprise mutation Y349C, and / or the truncated HC may be of isotype subclass IgG1 and comprise mutation S354C, and residues are numbered according to the Eu system.

[0025] In some embodiments, the monovalent antibody described herein comprises: a) a single-chain variable fragment (scFv) comprising the VH and VL linked to an Fc monomer; and b) a truncated HC lacking the variable and CH1 domains; wherein the Fc monomer linked to the scFv and the truncated HC are capable of dimerization. In certain embodiments, the Fc monomer linked to the scFv and the truncated HC comprise knobs-in-holes modifications, for example, wherein the Fc monomer linked to the scFv is of isotype subclass IgG1 and comprises mutations T366S, L368A, and Y407A in the CH3 domain, and the truncated HC is of isotype subclass IgG1 and comprises mutation T366W in the CH3 domain, with residues numbered according to the Eu system. Additionally or alternatively, the Fc monomer linked to the scFv may be of isotype subclass IgG1 and comprise mutation Y349C, and / or the truncated HC may be of isotype subclass IgG1 and comprise mutation S354C, with residues numbered according to the Eu system.

[0026] In certain embodiments, the anti-TNFα antibodies or antigen-binding portions described herein have a lower binding affinity for human TNFα at pH 6.0 than at pH 7.4. Compared to antibodies comprising the VH and VL amino acid sequences of SEQ ID NOs: 2 and 4, respectively; SEQ ID NOs: 6 and 8, respectively; SEQ ID NOs: 10 and 12, respectively; or SEQ ID NOs: 14 and 16, respectively, the antibodies or portions thereof may be less degraded in vivo, more recycled to the cell surface in vivo, have a longer half-life in vivo, be less immunogenic in vivo, or any combination thereof. In certain embodiments, the antibodies or antigen-binding portions do not form large immune complexes.

[0027] The present invention also provides bispecific binding molecules having the binding specificity of an anti-TNFα antibody of the invention and the binding specificity of a second, distinct antibody. In some embodiments, the second antibody is an anti-IL17A antibody, an anti-IL23 antibody, or an anti-Angiopoietin 2 (Ang2) antibody.

[0028] The present disclosure also provides immunoconjugates comprising an anti-TNFα antibody or antigen-binding portion of the present disclosure linked to a therapeutic agent. In some embodiments, the therapeutic agent is an anti-inflammatory agent or an immunosuppressant, such as a steroid.

[0029] The present invention also provides isolated nucleic acid molecules comprising nucleotide sequences encoding the heavy and light chain sequences of an anti-TNFα antibody or antigen-binding portion thereof of the present invention. In some embodiments, the isolated nucleic acid molecule comprises the following nucleotide sequences: a) SEQ ID NOs: 29 and 15; b) SEQ ID NOs: 31 and 15; c) SEQ ID NOs: 33 and 15; d) SEQ ID NOs: 35 and 37; e) SEQ ID NOs: 13 and 39; f) SEQ ID NOs: 13 and 41; g) SEQ ID NOs: 13 and 43; h) SEQ ID NOs: 13 and 45; i) SEQ ID NOs: 13 and 47; j) SEQ ID NOs: 29 and 39; k) SEQ ID NOs: 29 and 41; l) SEQ ID NOs: 29 and 43; m) SEQ ID NOs: 29 and 45; n) SEQ ID NOs: 31 and 39; o) SEQ ID NOs: 31 and 41; p) SEQ ID NOs: 31 and 43; q) SEQ ID NOs: 31 and 45; r) SEQ ID NOs: 33 and 39; s) SEQ ID NOs: 33 and 41; t) SEQ ID NOs: 33 and 43; u) SEQ ID NOs: 33 and 45; v) SEQ ID NOs: 49 and 7; w) SEQ ID NOs: 51 and 7; x) SEQ ID NOs: 53 and 7; y) SEQ ID NOs: 5 and 55; z) SEQ ID NOs: 5 and 57; aa) SEQ ID NOs: 5 and 59; bb) SEQ ID NOs: 5 and 61; cc) SEQ ID NOs: 5 and 63; dd) SEQ ID NOs: 5 and 65; ee) SEQ ID NOs: 5 and 67; ff) SEQ ID NOs: 5 and 69; gg) SEQ ID NOs: 5 and 71; or hh) SEQ ID NOs: 5 and 73.

[0030] Also provided is a vector(s) comprising the isolated nucleic acid molecule(s), wherein said vector further comprises expression control sequence(s) operably linked to the isolated nucleic acid molecule(s).

[0031] The present invention also provides host cells comprising a nucleotide sequence encoding the heavy chain sequence and a nucleotide sequence encoding the light chain sequence of an anti-TNFα antibody or antigen-binding portion of the invention. In some embodiments, the host cell comprises a nucleotide sequence selected from a) through hh) above. Also provided are methods for producing an anti-TNFα antibody or antigen-binding portion thereof, comprising providing a host cell, culturing the host cell under conditions suitable for expression of the antibody or antigen-binding portion, and isolating the resulting antibody or antigen-binding portion.

[0032] The invention also provides pharmaceutical compositions comprising an anti-TNFα antibody or antigen-binding portion thereof, a bispecific binding molecule thereof, or an immunoconjugate thereof, and a pharmaceutically acceptable excipient.

[0033] The present invention also provides a method for treating an autoimmune or inflammatory disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-TNFα antibody or antigen-binding portion thereof, a bispecific binding molecule thereof, or an immunoconjugate thereof.

[0034] The invention also provides use of an anti-TNFα antibody or antigen-binding portion thereof, a bispecific binding molecule thereof, or an immunoconjugate thereof for the manufacture of a medicament for treating an autoimmune or inflammatory disease in a patient in need thereof.

[0035] The invention also provides an anti-TNFα antibody or antigen-binding portion thereof, a bispecific binding molecule thereof, or an immunoconjugate thereof for use in treating an autoimmune or inflammatory disease in a patient in need thereof.

[0036] In some embodiments, the autoimmune or inflammatory disease is rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, ankylosing spondylitis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, hidradenitis suppurativa, polyarticular juvenile idiopathic arthritis, panuveitis, or Alzheimer's disease. In some embodiments, the patient is treated with an additional therapeutic agent, e.g., an anti-inflammatory or immunosuppressant drug such as methotrexate.

[0037] The present invention also provides kits comprising an anti-TNFα antibody or antigen-binding portion thereof, a bispecific binding molecule thereof, or an immunoconjugate thereof, hi some embodiments, the kits are for use in the treatments described herein.

[0038] The invention also provides an article of manufacture comprising an anti-TNFα antibody or antigen-binding portion thereof, a bispecific binding molecule thereof, or an immunoconjugate thereof, wherein the article of manufacture is suitable for treating an autoimmune or inflammatory disease in a patient in need thereof. In some embodiments, the treatment is a treatment described herein.

[0039] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description, while illustrating aspects of the present invention, is given by way of example only and is not intended to be limiting. Various changes and modifications within the scope of the present invention may become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0040] [Figure 1]Figure 1 is a graph showing the binding of high-affinity anti-TNFα Fabs to biotinylated TNFα. Bacterially expressed Fabs were captured on ELISA plates and then titrated with biotinylated human TNFα. Binding of biotinylated TNFα to various Fabs was quantified after extended dissociation at pH 7.4 in the presence of 100 nM unlabeled TNFα. Mutants A1, cb1-3, 4.2a-6, and Ab4 all bound more tightly than Ab1 Fab. [Figure 2] Figure 2 is a pair of graphs showing the pH sensitivity of binding of a high-affinity anti-TNFα Fab. Binding of the Fab to immobilized human TNFα was quantified after extended dissociation at pH 7.4 (panel A) or pH 6.0 (panel B) in the presence of 100 nM soluble TNFα. [Figure 3] Figure 3 is a pair of graphs showing the pH sensitivity of binding of 4.2a-6 template Fab variants with CDR histidine mutations. Binding of the Fab variants to immobilized human TNFα was quantified after extended dissociation at pH 7.4 (panel A) or pH 6.0 (panel B) in the presence of 100 nM soluble TNFα. [Figure 4] Figure 4 is a set of graphs showing the pH sensitivity of binding of 4.2a-6 template Fab variants with combined CDR histidine mutations. Binding of the Fab variants to immobilized human TNFα was quantified after extended dissociation at pH 7.4 (panels A and C) or pH 6.0 (panels B and D) in the presence of 100 nM soluble TNFα. [Figure 5] Figure 5 is a set of graphs depicting the pH sensitivity of binding of A1 template Fab variants with CDR histidine mutations. Binding of the Fab variants to immobilized human TNFα was quantified after extended dissociation at pH 7.4 (panels A and C) or pH 6.0 (panels B and D) in the presence of 100 nM soluble TNFα. [Figure 6]Figure 6 is a pair of graphs showing the pH sensitivity of binding of selected A1 template Fab variants with CDR histidine mutations. Binding of the Fab variants to immobilized human TNFα was quantified after extended dissociation at pH 7.4 (panel A) or pH 6.0 (panel B) in the presence of 100 nM soluble TNFα. [Figure 7] Figure 7 is a pair of graphs showing the pH sensitivity of binding of selected A1 template or selected 4.2a-6 template Fab variants with CDR histidine mutations. Binding of the Fab variants to soluble biotinylated mouse TNFα was quantified in the presence of 100 nM soluble TNFα after extended dissociation at pH 7.4 (panel A) or pH 6.0 (panel B). DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description of the Invention The present invention provides novel anti-human TNFα antibodies and antigen-binding portions thereof that can be used to treat autoimmune and / or inflammatory diseases. Unless otherwise specified, "TNFα" as used herein refers to human TNFα. The sequence of the human TNFα polypeptide is shown below: TIFF2025526279000001.tif41164

[0042] As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) can refer to a tetramer comprising two heavy (H) chains (approximately 50-70 kDa) and two light (L) chains (approximately 25 kDa) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into regions of hypervariability called "complementarity-determining regions" (CDRs) and highly conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDRs (herein, H-CDR refers to a CDR derived from the heavy chain, and L-CDR refers to a CDR derived from the light chain) and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the antibodies described herein may be bivalent antibodies. As used herein, the term "bivalent antibody" refers to an antibody having two antigen-binding sites. In some embodiments, the antibodies described herein may be monovalent antibodies comprising less than two HCs and two LCs (e.g., comprising a single VH and VL, or HC and LC, from an anti-TNFα antibody). As used herein, the term "monovalent antibody" refers to an antibody having one antigen-binding site.

[0043] The amino acid numbering and / or assignment of FR and CDR regions in the heavy or light chains can be found in IMGT (登録商標)definition (Lefranc et al., Dev Comp Immunol. (2003) 27(1):55-77), Eu numbering, or Kabat definition, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991); Chothia & Lesk, J Mol Biol. (1987) 196:901-17; Chothia et al., Nature (1989) 342:878-83; MacCallum et al., J Mol Biol. (1996) 262:732-45; or Honegger and Plueckthun, J Mol Biol. (2001) 309(3):657-70 (“AHo” numbering).

[0044] In some embodiments, the antibodies or antigen-binding portions thereof of the present invention are isolated antibodies or antigen-binding portions. The term "isolated protein," "isolated polypeptide," or "isolated antibody" refers to a protein, polypeptide, or antibody that, by virtue of its origin or source of derivation, (1) is not associated with naturally associated components with which it is naturally associated; (2) is free from other proteins from the same species; (3) is expressed by cells of a different species; and / or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the cell in which it is naturally derived would be "isolated" from naturally associated components. A protein can also be rendered substantially free of naturally occurring components by isolation using protein purification techniques well known in the art.

[0045] The term "affinity" refers to a measure of the binding strength between an antigen and an antibody or antigen-binding fragment thereof, or related molecule such as a bispecific binding molecule. The intrinsic avidity of an antibody for an antigen is usually measured using the binding affinity equilibrium constant (K D ) The antibody or antigen-binding portion is expressed as KD When the K is 1 μM or less, e.g., 100 nM or less or 10 nM or less, it is said to specifically bind to the antigen. D Binding affinity constants can be measured using, for example, the IBIS MX96 SPR system from IBIS Technologies, the Carterra LSA SPR platform, or the Octet (商標) Surface plasmon resonance (BIAcore) system (商標) ) or biolayer interferometry.

[0046] As used herein, the term "epitope" refers to a portion (determinant) of an antigen that specifically binds to an antibody or its antigen-binding portion. Epitopes generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be "linear" or "three-dimensional." In a linear epitope, all of the points of interaction between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a three-dimensional epitope, multiple points of interaction occur between amino acid residues on the protein that are separated from one another in the primary amino acid sequence. Once a desired epitope on an antigen has been determined, antibodies to that epitope can be generated using techniques well known in the art. For example, antibodies to a linear epitope can be generated by immunizing an animal with a peptide containing the amino acid residues of the linear epitope. Antibodies to conformational epitopes can be generated, for example, by immunizing animals with a minidomain containing the relevant amino acid residues of the conformational epitope. Antibodies to specific epitopes can also be generated, for example, by immunizing animals with a target molecule of interest (e.g., TNFα) or a relevant portion thereof and screening for binding to the epitope.

[0047] Whether an antibody binds to the same epitope of TNFα or competes for binding with the antibodies described herein can be determined using methods known in the art, including, but not limited to, competition assays, epitope binning, and alanine scanning. In some embodiments, the antibodies described herein are allowed to bind to TNFα under saturating conditions, and then the ability of the test antibody to bind to the antigen is measured. If the test antibody can bind to the antigen simultaneously with the reference antibody, the test antibody binds to a different epitope than the reference antibody. However, if the test antibody cannot simultaneously bind to the antigen, the test antibody binds to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope bound by the antibodies described herein. This experiment can be performed using, for example, ELISA, RIA, BIACORE. (商標) , SPR, Bio-Layer Interferometry, flow cytometry, etc. To determine whether the antibodies described herein cross-compete with other antibodies for binding to TNFα, the competition method described above can be used in two directions: to determine whether a known antibody blocks the test antibody, and vice versa. Such cross-competition experiments can be performed, for example, on an IBIS MX96 SPR instrument or an Octet (商標) This can be done using the system.

[0048] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human TNFα or a portion thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" include: (i) a Fab fragment: a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment: (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain; and (vi) an isolated complementarity-determining region (CDR) capable of specifically binding to an antigen. Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be recombinantly linked by a synthetic linker, allowing the VL and VH domains to pair and be produced as a single protein chain to form a monovalent molecule known as a single-chain variable fragment (scFv). Antigen-binding molecules comprising VH and / or VL are also contemplated by the present invention. In the case of VH, the molecule may include one or more of the CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to pair with complementary domains on another chain to form two antigen-binding sites. The present invention also contemplates antigen-binding portions of the anti-TNFα antibodies described herein, where the antigen-binding portion retains a functional property of the cognate antibody, and such antigen-binding portions can be used wherever the cognate antibody is used.

[0049] Anti-TNFα antibody The present invention provides novel therapeutic anti-TNFα antibodies engineered to be less immunogenic. Such engineered antibodies may maintain more stable serum antibody levels and have a greater or longer therapeutic effect than the parent antibody. In some embodiments, the antibodies of the present invention are engineered to prevent the formation of large immune complexes (ICs), enhance dissociation from TNFα at acidic pH, or both. As used herein, "large ICs" refers to immune complexes consisting of two or more TNFα trimers and three or more antibodies or antigen-binding moieties. In certain embodiments, the antibodies possess a pH-sensitive antigen-binding function ("pH switch"). This pH switch allows the antibodies to bind and neutralize serum (soluble) and membrane-bound TNFα at physiological pH (e.g., pH 7.4) while simultaneously allowing dissociation upon internalization into the acidic endosomal environment (e.g., pH 6.0). Degraded antibodies are recycled to the cell surface via FcRn, and antigens are delivered to lysosomes for degradation. In certain embodiments, the antibodies are monovalent. Without being bound by theory, it is postulated that monovalency reduces or eliminates the formation of large ICs via antibody-mediated cross-linking of TNFα. In certain embodiments, the antibodies of the invention incorporate a pH switch and are monovalent.

[0050] In some embodiments, the anti-TNFα antibodies, or antigen-binding portions thereof, of the invention are derived from high-affinity variants of the parent anti-TNFα antibody “Ab1” comprising the amino acid sequence shown below (variable domains are italicized, CDRs are underlined): TIFF2025526279000002.tif79164

[0051] In some embodiments, a high affinity variant of Ab1 comprises: a) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8 (“A1”); b) a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 12 (“cb1-3”); or c) VH comprising the amino acid sequence of SEQ ID NO: 14 and VL comprising the amino acid sequence of SEQ ID NO: 16 ("4.2a-6").

[0052] In some embodiments, the anti-TNFα antibody or antigen-binding portion thereof of the present invention binds to the same epitope of human TNFα as the reference high-affinity variant and comprises a VH and a VL that are at least 90% identical to the VH and VL, respectively, of the reference high-affinity variant. In some embodiments, the anti-TNFα antibody or antigen-binding portion has VH and VL amino acid sequences that contain a total of at least one, two, three, four, or five amino acid substitutions from the VH and VL amino acid sequences of the reference high-affinity variant. In certain embodiments, the VH and VL amino acid sequences contain a total of one amino acid substitution from the VH and VL amino acid sequences of the reference high-affinity variant. In certain embodiments, the VH and VL amino acid sequences contain a total of two amino acid substitutions from the VH and VL amino acid sequences of the reference high-affinity variant. In certain embodiments, the VH and VL amino acid sequences contain a total of three amino acid substitutions from the VH and VL amino acid sequences of the reference high-affinity variant. In certain embodiments, the VH and VL amino acid sequences contain a total of four amino acid substitutions from the VH and VL amino acid sequences of the reference high-affinity variant. In certain embodiments, the VH and VL amino acid sequences contain a total of five amino acid substitutions from the VH and VL amino acid sequences of the reference high-affinity variant. In certain embodiments, the amino acid substitutions may alter the binding affinity of an antibody or moiety at a particular pH value. For example, an engineered antibody or moiety may have a lower binding affinity for TNFα at low pH (e.g., pH 6.0) compared to high pH (e.g., pH 7.4). In some embodiments, the EC50 for binding at lower pH may be increased by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 50-fold, 75-fold, 100-fold, 500-fold, 1000-fold, 2000-fold, or 4000-fold compared to the binding affinity at higher pH.

[0053] In some embodiments, the amino acid substitution(s) are in the FR, or the FR and the CDR of the anti-TNFα antibody or antigen-binding portion. In some embodiments, the amino acid substitution(s) are in the CDR of the anti-TNFα antibody or antigen-binding portion. In certain embodiments, the amino acid substitution(s) are in the H-CDR3, L-CDR1, L-CDR3, or any combination thereof (e.g., L-CDR1 and L-CDR3, H-CDR3 and L-CDR1, H-CDR3 and L-CDR3, or H-CDR3, L-CDR1 and L-CDR3). The CDRs are determined by the Kabat method, the Chothia method, the IMGT method, the contact method, the AHo method, or a combination thereof. In certain embodiments, the CDRs are determined as shown in the Ab1 sequence above (SEQ ID NOs: 120 and 121).

[0054] In certain embodiments, the anti-TNFα antibody or portion is - H-CDR1 comprising a sequence selected from SEQ ID NOs: 75, 81 and 87; - H-CDR2 comprising SEQ ID NO: 76; - H-CDR3 comprising a sequence selected from SEQ ID NOs: 77, 82, 97, 98, 101, 102, 109, 110 and 111; - L-CDR1 comprising a sequence selected from SEQ ID NOs: 78, 83, 84, 88, 104, 107, 112, 114, 117, 118, and 119; - L-CDR2 comprising a sequence selected from SEQ ID NOs: 79, 85 and 89; and - an L-CDR3 comprising a sequence selected from SEQ ID NOs: 80, 86, 90, 99, 100, 103, 105, 106, 108, 113, 115 and 116; Including, wherein said antibody or portion does not comprise the six CDR sequences of SEQ ID NOs: 75, 76, 77, 78, 79 and 80; SEQ ID NOs: 81, 76, 82, 83, 79 and 80; SEQ ID NOs: 81, 76, 77, 84, 85 and 86; or SEQ ID NOs: 87, 76, 77, 88, 89 and 90.

[0055] In certain embodiments, the anti-TNFα antibody or portion is - a VH comprising a sequence selected from SEQ ID NOs: 2, 6, 10, 14, 30, 32, 34, 36, 50, 52 and 54, or a sequence at least 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and - a VL comprising a sequence selected from SEQ ID NOs: 4, 8, 12, 16, 38, 40, 42, 44, 46, 48, 56, 58, 60, 62, 64, 66, 68, 70, 72 and 74, or a sequence at least 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; Including, wherein the antibody or portion thereof does not comprise the VH and VL sequences of SEQ ID NOs: 2 and 4, the VH and VL sequences of SEQ ID NOs: 6 and 8, the VH and VL sequences of SEQ ID NOs: 10 and 12, or the VH and VL sequences of SEQ ID NOs: 14 and 16.

[0056] In certain embodiments, the anti-TNFα antibody or portion is - H-CDR1 comprising SEQ ID NO: 87; - H-CDR2 comprising SEQ ID NO: 76; - H-CDR3 comprising a sequence selected from SEQ ID NOs: 77, 97, 98, 101 and 102; - L-CDR1 comprising a sequence selected from SEQ ID NOs: 88, 104, 107; - L-CDR2 comprising SEQ ID NO: 89; and - an L-CDR3 comprising a sequence selected from SEQ ID NOs: 90, 103, 105, 106 and 108; Including, wherein said antibody or portion thereof does not comprise the H-CDR1-3 and L-CDR1-3 sequences of SEQ ID NOs: 87, 76, 77, 88, 89, and 90.

[0057] In certain embodiments, the anti-TNFα antibody or portion is -SEQ ID NOs: 87, 76 and 77, respectively; -SEQ ID NOs: 87, 76 and 97, respectively; -SEQ ID NOs: 87, 76 and 98, respectively; SEQ ID NOs: 87, 76, and 101, respectively; or SEQ ID NOs: 87, 76, and 102, respectively and an H-CDR1-3 sequence comprising: -SEQ ID NOs: 88, 89 and 90, respectively; -SEQ ID NOs: 88, 89 and 103, respectively; -SEQ ID NOs: 104, 89 and 90, respectively; -SEQ ID NOs: 88, 89 and 105, respectively; -SEQ ID NOs: 88, 89 and 106, respectively; SEQ ID NOs: 107, 89 and 90, respectively; or SEQ ID NOs: 104, 89 and 108, respectively an L-CDR1-3 sequence comprising wherein said antibody or portion does not comprise the H-CDR1-3 and L-CDR1-3 sequences of SEQ ID NOs: 87, 76, 77, 88, 89, and 90, respectively.

[0058] In certain embodiments, the anti-TNFα antibody or portion is - SEQ ID NOs: 87, 76, 97, 88, 89 and 90, respectively; - SEQ ID NOs: 87, 76, 98, 88, 89 and 90, respectively; - SEQ ID NOs: 87, 76, 101, 88, 89 and 90, respectively; - SEQ ID NOs: 87, 76, 102, 88, 89 and 103, respectively; - SEQ ID NOs: 87, 76, 77, 104, 89 and 90, respectively; -SEQ ID NOs: 87, 76, 77, 88, 89 and 105, respectively; -SEQ ID NOs: 87, 76, 77, 88, 89 and 106, respectively; -SEQ ID NOs: 87, 76, 77, 107, 89 and 90, respectively; - SEQ ID NOs: 87, 76, 77, 104, 89 and 108, respectively; -SEQ ID NOs: 87, 76, 97, 104, 89 and 90, respectively; -SEQ ID NOs: 87, 76, 97, 88, 89 and 105, respectively; -SEQ ID NOs: 87, 76, 97, 88, 89 and 106, respectively; -SEQ ID NOs: 87, 76, 97, 107, 89 and 90, respectively; -SEQ ID NOs: 87, 76, 98, 104, 89 and 90, respectively; -SEQ ID NOs: 87, 76, 98, 88, 89 and 105, respectively; -SEQ ID NOs: 87, 76, 98, 88, 89 and 106, respectively; -SEQ ID NOs: 87, 76, 98, 107, 89 and 90, respectively; - SEQ ID NOs: 87, 76, 101, 104, 89 and 90, respectively; - SEQ ID NOs: 87, 76, 101, 88, 89 and 105, respectively; - SEQ ID NOs: 87, 76, 101, 88, 89 and 106, respectively; or - SEQ ID NOs: 87, 76, 101, 107, 89 and 90, respectively The H-CDR1-3 and L-CDR1-3 sequences include:

[0059] In certain embodiments, the anti-TNFα antibody or portion comprises a VH comprising a sequence selected from SEQ ID NOs: 14, 30, 32, 34, and 36, or a sequence at least 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a VL comprising a sequence selected from SEQ ID NOs: 16, 38, 40, 42, 44, 46, and 48, or a sequence at least 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. wherein the antibody or portion does not comprise the VH and VL sequences of SEQ ID NOs: 14 and 16, respectively.

[0060] In certain embodiments, the anti-TNFα antibody or portion is -SEQ ID NOs: 30 and 16, respectively; -SEQ ID NOs: 32 and 16, respectively; -SEQ ID NOs: 34 and 16, respectively; - SEQ ID NOs: 36 and 38, respectively; -SEQ ID NOs: 14 and 40, respectively; -SEQ ID NOs: 14 and 42, respectively; -SEQ ID NOs: 14 and 44, respectively; -SEQ ID NOs: 14 and 46, respectively; -SEQ ID NOs: 14 and 48, respectively; - SEQ ID NOs: 30 and 40, respectively; - SEQ ID NOs: 30 and 42, respectively; -SEQ ID NOs: 30 and 44, respectively; -SEQ ID NOs: 30 and 46, respectively; - SEQ ID NOs: 32 and 40, respectively; -SEQ ID NOs: 32 and 42, respectively; -SEQ ID NOs: 32 and 44, respectively; -SEQ ID NOs: 32 and 46, respectively; - SEQ ID NOs: 34 and 40, respectively; - SEQ ID NOs: 34 and 42, respectively; SEQ ID NOs: 34 and 44, respectively; or SEQ ID NOs: 34 and 46, respectively. and VH and VL comprising the sequence:

[0061] In certain embodiments, an anti-TNFα antibody or portion thereof is - H-CDR1 comprising SEQ ID NO: 81; - H-CDR2 comprising SEQ ID NO: 76; - H-CDR3 comprising a sequence selected from SEQ ID NOs: 82, 109, 110, and 111; - L-CDR1 comprising a sequence selected from SEQ ID NOs: 83, 112, 114, 117, 118, and 119; - L-CDR2 consisting of SEQ ID NO: 79; and - L-CDR3 comprising a sequence selected from SEQ ID NOs: 80, 99, 100, 113, 115, and 116 wherein the antibody or portion thereof does not comprise the H-CDR1-3 and L-CDR1-3 sequences of SEQ ID NOs: 81, 76, 82, 83, 79 and 80, respectively.

[0062] In certain embodiments, the anti-TNFα antibody or portion is -SEQ ID NOs: 81, 76 and 82, respectively; -SEQ ID NOs: 81, 76 and 109, respectively; SEQ ID NOs: 81, 76 and 110, respectively; or SEQ ID NOs: 81, 76 and 111, respectively and an H-CDR1-3 sequence comprising: -SEQ ID NOs: 83, 79 and 80, respectively; -SEQ ID NOs: 112, 79 and 80, respectively; -SEQ ID NOs: 83, 79 and 99, respectively; -SEQ ID NOs: 83, 79 and 113, respectively; - SEQ ID NOs: 83, 79 and 100, respectively; -SEQ ID NOs: 114, 79 and 80, respectively; -SEQ ID NOs: 83, 79 and 115, respectively; -SEQ ID NOs: 112, 79 and 116, respectively; -SEQ ID NOs: 117, 79 and 80, respectively; SEQ ID NOs: 118, 79 and 80, respectively; or -SEQ ID NOs: 119, 79 and 80, respectively; L-CDR1-3 sequence containing wherein the antibody or portion thereof does not comprise the H-CDR1-3 and L-CDR1-3 sequences of SEQ ID NOs: 81, 76, 82, 83, 79 and 80, respectively.

[0063] In certain embodiments, the anti-TNFα antibody or portion is - SEQ ID NOs: 81, 76, 109, 83, 79 and 80, respectively; -SEQ ID NOs: 81, 76, 110, 83, 79 and 80, respectively; - SEQ ID NOs: 81, 76, 111, 83, 79 and 80, respectively; -SEQ ID NOs: 81, 76, 82, 112, 79 and 80, respectively; -SEQ ID NOs: 81, 76, 82, 83, 79 and 99, respectively; -SEQ ID NOs: 81, 76, 82, 83, 79 and 113, respectively; - SEQ ID NOs: 81, 76, 82, 83, 79 and 100, respectively; -SEQ ID NOs: 81, 76, 82, 114, 79 and 80, respectively; -SEQ ID NOs: 81, 76, 82, 83, 79 and 115, respectively; -SEQ ID NOs: 81, 76, 82, 112, 79 and 116, respectively; -SEQ ID NOs: 81, 76, 82, 117, 79 and 80, respectively; SEQ ID NOs: 81, 76, 82, 118, 79 and 80, respectively; or - SEQ ID NOs: 81, 76, 82, 119, 79 and 80, respectively The H-CDR1-3 and L-CDR1-3 sequences include:

[0064] In certain embodiments, the anti-TNFα antibody or portion comprises a VH comprising a sequence selected from SEQ ID NOs: 6, 50, 52, and 54, or a sequence at least 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a VL comprising a sequence selected from SEQ ID NOs: 8, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, or a sequence at least 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, wherein the antibody or portion does not comprise the VH and VL sequences of SEQ ID NOs: 6 and 8, respectively.

[0065] In certain embodiments, the anti-TNFα antibody or portion is -SEQ ID NOs: 50 and 8, respectively; - SEQ ID NOs: 52 and 8, respectively; - SEQ ID NOs: 54 and 8, respectively; -SEQ ID NOs: 6 and 56, respectively; -SEQ ID NOs: 6 and 58, respectively; -SEQ ID NOs: 6 and 60, respectively; -SEQ ID NOs: 6 and 62, respectively; -SEQ ID NOs: 6 and 64, respectively; -SEQ ID NOs: 6 and 66, respectively; -SEQ ID NOs: 6 and 68, respectively; -SEQ ID NOs: 6 and 70, respectively; SEQ ID NOs: 6 and 72, respectively; or SEQ ID NOs: 6 and 74, respectively and VH and VL comprising the sequence:

[0066] The anti-TNFα antibodies described herein can be IgG, IgM, IgE, IgA, or IgD molecules, but are typically of the IgG isotype, e.g., IgG subclasses IgG1, IgG2a or IgG2b, IgG3, or IgG4. In certain embodiments, the antibodies are of the isotype subclass IgG1.

[0067] The anti-TNFα antibodies described herein may be in a monovalent / monomeric form. Examples of such forms include any form comprising a single antigen-binding domain (e.g., a single VH / VL pair), including Fab, scFv, single-domain antibodies, VHH / nanobodies, UniDabs, VNARs, etc. Also contemplated are monovalent binding molecule forms such as adnexins, affibodies, affilins, anticalins, avimers, DARPins, etc., which have the binding specificity of the anti-TNFα antibodies described herein. The monovalent antibodies described herein may also include a constant (Fc) region component (e.g., a complete Fc region) that provides effector function (e.g., complete effector function).

[0068] In certain embodiments, the anti-TNFα antibodies described herein comprise an antigen-binding protein, which can be monovalent, bivalent, or multivalent. In some embodiments, the antigen-binding protein is monovalent (also referred to herein as "Fab") and comprises the VH and VL, or HC and LC, of an anti-TNFα antibody described herein. In certain embodiments, the antigen-binding protein is monovalent and comprises the HC and LC of an anti-TNFα antibody described herein. In some embodiments, the monovalent anti-TNFα antibodies described herein are heterotrimers comprising an antibody HC that binds to the antibody LC to form the antigen-binding domain, and the antibody HC dimerizes with a polypeptide that is a "truncated heavy chain" (i.e., an HC lacking the variable and CH1 domains) to form an Fc domain. The truncated heavy chain comprises or consists of an Fc monomer (i.e., one of the two polypeptides that dimerize to form the Fc domain). In certain embodiments, the Fc monomer comprises the CH2 and CH3 of an antibody heavy chain, such as an IgG heavy chain, where the IgG may be IgG1, IgG2, IgG2, or IgG4. In certain embodiments, dimerization between the antibody HC and a truncated HC provides a fully functional Fc domain, and can maintain the pharmacokinetic and effector function properties of the parent antibody (e.g., Ab1 or a high-affinity variant thereof described herein).

[0069] In certain embodiments, the monovalent anti-TNFα antibodies described herein comprise an scFv. In certain embodiments, the scFv comprises the VH and VL of an anti-TNFα antibody described herein. In certain embodiments, the monovalent anti-TNFα antibodies described herein are heterodimers (e.g., a single chain comprising an scFv and an Fc monomer of an anti-TNFα antibody described herein, and an additional (truncated) HC lacking the variable domain and the CH1 domain (e.g., a constant domain fragment such as an Fc monomer)). The single chain can be configured, for example, as a VL-linker-VH-Fc monomer. In certain embodiments, dimerization between the Fc monomer portion of the single chain and the Fc monomer portion of the additional HC provides a fully functional Fc domain and can maintain the pharmacokinetic and effector function properties of the parent antibody (e.g., Ab1 or a high-affinity variant thereof described herein).

[0070] In certain embodiments of monovalent anti-TNFα antibody heterotrimers or heterodimers, the heavy chain Fc heterodimer is in a form described, for example, in Brinkmann and Kontermann, MAbs 9:182-212 (2017). For example, "knobs-in-holes," HA-TF, ZW1, CH3 charge pair, EW-RVT, LUZ-Y, strand exchange engineered domain (SEEDbody), Biclonic, DuoBody, BEAT, 7.8.60, 20.8.34, Triomab / Quadroma, or CrossMAb strategies can be used to promote heterodimerization (e.g., more than homodimerization) of antibody heavy chain Fc monomers and truncated heavy chain Fc monomers. In certain embodiments, a "knob-in-hole" approach may be used, where "knob" variants of a domain are obtained by replacing an amino acid with a small side chain (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine) with another amino acid with a larger side chain (e.g., arginine, phenylalanine, tyrosine, tryptophan, etc.). "Hole" variants of a domain are obtained by replacing an amino acid with a large side chain (e.g., arginine, phenylalanine, tyrosine, tryptophan, etc.) with another amino acid with a small side chain (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine). In certain embodiments, the knob and / or hole mutations are in the CH3 domain. In certain embodiments, both Fc monomers are derived from IgG1, and the antibody heavy chain Fc monomer may contain the mutations T366S, L368A, and Y407A in the CH3 domain, and the truncated heavy chain Fc monomer may contain the mutation T366W in the CH3 domain, or vice versa, with residues numbered according to the Eu system. Additionally or alternatively, the antibody heavy chain Fc monomer may contain the mutation Y349C, and the truncated heavy chain Fc monomer may contain the mutation S354C, or vice versa, with residues numbered according to the Eu system.

[0071] In some embodiments, the present invention provides an anti-TNFα antibody or antigen-binding portion thereof (e.g., a monovalent anti-TNFα antibody or antigen-binding portion thereof), wherein the antibody comprises H-CDR1-3 and L-CDR1-3 comprising SEQ ID NOs: 81, 76, 109, 83, 79, and 80, respectively. In some embodiments, the antibody or portion comprises a VH comprising SEQ ID NO: 50 and a VL comprising SEQ ID NO: 8. In particular embodiments, the antibody is monovalent and comprises a monovalent antigen binding protein comprising an HC and LC having said VH and VL, respectively, and a truncated HC lacking the variable domain and the CH1 domain, wherein said antigen binding protein HC and truncated HC are capable of dimerizing.

[0072] In some embodiments, the present invention provides an anti-TNFα antibody or antigen-binding portion thereof (e.g., a monovalent anti-TNFα antibody or antigen-binding portion thereof), wherein the antibody comprises H-CDR1-3 and L-CDR1-3 comprising SEQ ID NOs: 81, 76, 111, 83, 79, and 80, respectively. In some embodiments, the antibody or portion comprises a VH comprising SEQ ID NO: 54 and a VL comprising SEQ ID NO: 8. In particular embodiments, the antibody is monovalent and comprises a monovalent antigen binding protein comprising an HC and LC having said VH and VL, respectively, and a truncated HC lacking the variable domain and the CH1 domain, wherein the antigen binding protein HC and truncated HC are capable of dimerizing.

[0073] In some embodiments, the present invention provides an anti-TNFα antibody or antigen-binding portion thereof (e.g., a monovalent anti-TNFα antibody or antigen-binding portion thereof), wherein the antibody comprises H-CDR1-3 and L-CDR1-3 comprising SEQ ID NOs: 81, 76, 82, 112, 79, and 80, respectively. In some embodiments, the antibody or portion comprises a VH comprising SEQ ID NO: 6 and a VL comprising SEQ ID NO: 56. In particular embodiments, the antibody is monovalent and comprises a monovalent antigen binding protein comprising an HC and LC having said VH and VL, respectively, and a truncated HC lacking the variable domain and the CH1 domain, wherein the antigen binding protein HC and truncated HC are capable of dimerizing.

[0074] In some embodiments, the present invention provides an anti-TNFα antibody or antigen-binding portion thereof (e.g., a monovalent anti-TNFα antibody or antigen-binding portion thereof), wherein the antibody comprises H-CDR1-3 and L-CDR1-3 comprising SEQ ID NOs: 81, 76, 82, 114, 79, and 80, respectively. In some embodiments, the antibody or portion comprises a VH comprising SEQ ID NO: 6 and a VL comprising SEQ ID NO: 64. In particular embodiments, the antibody is monovalent and comprises a monovalent antigen binding protein comprising an HC and LC having said VH and VL, respectively, and a truncated HC lacking the variable domain and the CH1 domain, wherein the antigen binding protein HC and truncated HC are capable of dimerizing.

[0075] In some embodiments, the present invention provides an anti-TNFα antibody or antigen-binding portion thereof (e.g., a monovalent anti-TNFα antibody or antigen-binding portion thereof), wherein the antibody comprises H-CDR1-3 and L-CDR1-3 comprising SEQ ID NOs: 81, 76, 82, 112, 79, and 116, respectively. In some embodiments, the antibody or portion comprises a VH comprising SEQ ID NO: 6 and a VL comprising SEQ ID NO: 68. In particular embodiments, the antibody is monovalent and comprises a monovalent antigen-binding protein comprising an HC and LC having said VH and VL, respectively, and a truncated HC lacking the variable domain and the CH1 domain, wherein the Fab HC and truncated HC are capable of dimerizing.

[0076] In some embodiments, the invention provides an anti-TNFα antibody or antigen-binding portion thereof (e.g., a monovalent anti-TNFα antibody or antigen-binding portion thereof), wherein the antibody comprises H-CDR1-3 and L-CDR1-3 comprising SEQ ID NOs: 87, 76, 77, 88, 89, and 106, respectively. In some embodiments, the antibody or portion comprises a VH comprising SEQ ID NO: 14 and a VL comprising SEQ ID NO: 44. In particular embodiments, the antibody is monovalent and comprises a monovalent antigen-binding protein comprising an HC and LC having said VH and VL, respectively, and a truncated HC lacking the variable domain and the CH1 domain, wherein the Fab HC and truncated HC are capable of dimerizing.

[0077] In some embodiments, the constant region(s) of an anti-TNFα antibody or antigen-binding portion thereof described herein are mutated, e.g., to increase the effector function of the antibody or antigen-binding portion (e.g., Wang et al., Protein Cell (2018) 9(1):63-73; Kellner et al., Transfus Med Hemother. (2017) 44:327-36; or Robkopf et al., Antibodies (2020) 9(4):63). In certain embodiments, the mutations enhance ADCC or CDC. In some embodiments, the mutations are in IgG1 and include L235V, G236A, S239D, F243L, S267E, H268F, R292P, S298A, Y300L, V305I, S324T, N325S, K326W, L328F, A330L, I332E, E333A, E333S, K334A, P396L, or any combination thereof. For example, mutations can include F243L / R292P / Y300L / V305I / P396L; S239D / I332E; S239D / I332E / A330L; S298A / E333A / K334A; L235V / F143L / R292P / Y300L / P396L; G236A / S239D / I332E; K326W / E333S; S267E / H268F / S324T; S267E / H268F / S324T / G236A / I332E; S267E / L328F; or N325S / L328F. In some embodiments, the mutations may include L234Y / L235Q / G236W / S239M / H268D / D270E / S298A on one heavy chain and D270E / K32D / A330M / K334E on the other heavy chain.

[0078] Additionally or alternatively, the constant region(s) of the anti-TNFα antibodies or antigen-binding portions thereof described herein may be mutated to extend the half-life of the antibody or portion (e.g., Maeda et al., MAbs (2017) 9(5):844-53; Wang et al., supra; or as described in PCT Patent Publication WO 00 / 09560). In some embodiments, the mutations are in IgG1 and include M252Y, S254T, T256E, M428L, N434A, N434S, Y436T, Y436V, Q438R, S440E, or any combination thereof (Eu numbering). For example, mutations can include M252Y / S254T / T256E, M428L / N434S, N434A / Y436T / Q438R / S440E; N434A / Y436V / Q438R / S440E; M428L / N434A / Y436T / Q438R / S440E; M428L / N434A / Y436V / Q438R / S440E; or M428L / N434A / Q438R / S440E.

[0079] In some embodiments, the antibody is glycoengineered to enhance effector function (e.g., Li et al., Proc Natl Acad Sci USA (2017) 114(13):3485-90; or as described in Robkopf et al., supra). In certain embodiments, the antibody is glycoengineered to reduce fucose (e.g., defucosylated variants) or sialic acid content, or to enhance effector function (e.g., GlycoMAb variants). (商標) It is glycoengineered using technology.

[0080] In some embodiments, the framework or constant regions of the anti-TNFα antibodies, or antigen-binding portions thereof, described herein are mutated to alter the immunogenicity of the antibody and / or to provide sites for covalent or non-covalent binding to another molecule.

[0081] Combinations of any of the mutations described herein are also contemplated.

[0082] In some embodiments, the anti-TNFα antibodies or antigen-binding portions of the invention bind to human TNFα with an EC50 of 1e-007M, 5e-008M, 2e-008M, 1e-008M, 5e-009M, 2e-009M, 1e-009M, 5e-010M, 2e-010M, 1e-011M, 5e-011M, 2e-011M, 1e-011M, 5e-012M, 2e-012M, or 1e-012M or less, e.g., at pH 7.4. In certain embodiments, binding of the antibody or antigen-binding portion to human TNFα is reduced by at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 100-fold, 500-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, or 4000-fold at pH 6.0. In certain embodiments, the antibody or antigen-binding portion has a dissociation rate at pH 6.0 that is at least 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1500-fold, 2000-fold, or 2500-fold faster than the dissociation rate of an Ab1 or monovalent Ab1 described herein, or a high-affinity variant thereof. In some embodiments, the antibody or antigen-binding portion binds to human TNFα with an EC50 of 50 nM or less at pH 7.4 and has a dissociation rate for human TNFα at pH 6.0 that is at least 10-fold, 100-fold, or 1000-fold greater than the dissociation rate of an Ab1 or monovalent Ab1 described herein, or a high-affinity variant thereof. ... -1 In some embodiments, the antibody or antigen-binding portion binds to human TNFα at pH 7.4 with greater affinity than monovalent antibody AF-M2631 (comprising the VH and VL sequences of SEQ ID NOs: 22 and 4, respectively) and / or AF-M2637 (comprising the VH and VL sequences of SEQ ID NOs: 2 and 28, respectively). In particular embodiments, the antibody or antigen-binding portion binds to human TNFα at pH 7.4 with greater affinity than monovalent antibody AF-M2637.

[0083] In some embodiments, the anti-TNFα antibodies or antigen-binding portions of the invention bind to mouse TNFα with an EC50 of 1e-006M, 5e-007M, 1e-007M, 5e-008M, 2e-008M, 1e-008M, 5e-009M, 2e-009M, 1e-009M, 5e-010M, 2e-010M, 1e-011M, 5e-011M, 2e-011M, 1e-011M, 5e-012M, 2e-012M, or 1e-012M or less, e.g., at pH 7.4. In certain embodiments, binding of the antibody or antigen-binding portion to mouse TNFα is reduced at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 100-fold, 500-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, or 4000-fold at pH 6.0. In certain embodiments, the antibody or antigen-binding portion has a dissociation rate at pH 6.0 that is at least 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1500-fold, 2000-fold, or 2500-fold faster than the dissociation rate of Ab1 or a high affinity variant thereof described herein.

[0084] In certain embodiments, the anti-TNFα antibodies or antigen-binding portions of the invention bind to human and mouse TNFα with an EC50 of, e.g., 1e-008M, 5e-009M, 2e-009M, 1e-009M, 5e-010M, 2e-010M, 1e-011M, 5e-011M, 2e-011M, 1e-011M, 5e-012M, 2e-012M, or 1e-012M or less for each antigen, or any combination thereof, e.g., at pH 7.4.

[0085] In some embodiments, the anti-TNFα antibodies or antigen-binding portions of the invention have a longer half-life than Ab1 or its high affinity variants described herein, hi certain embodiments, the half-life may be at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, or 100-fold longer than the half-life of Ab1 or its high affinity variants described herein.

[0086] The present invention also contemplates anti-TNFα antibodies or antigen-binding portions having any combination of the above properties.

[0087] In some embodiments, an anti-TNFα antibody or antigen-binding portion of the invention has at least one (e.g., one, two, three, four, or five) of the following properties in any combination: -Does not form large immune complexes (i.e., two or more TNFα molecules bridged by three or more antibody molecules); - is less susceptible to in vivo degradation than Ab1 or its high affinity variants described herein; - Increased recycling to the cell surface in vivo compared to Ab1 or its high affinity variants described herein; - has a longer half-life than Ab1 or a high affinity variant thereof described herein; and - Less immunogenic in vivo than Ab1 or its high affinity variants described herein.

[0088] The anti-TNFα antibodies or antigen-binding portions of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibodies or portions thereof are derivatized so that TNFα binding is not adversely affected by the derivatization or labeling. Thus, the antibodies and antibody portions of the invention are intended to include both intact and modified forms of the anti-TNFα antibodies and antibody portions described herein. For example, the antibodies or antibody portions of the invention can be operably linked (by chemical conjugation, genetic fusion, non-covalent bonding, or other methods) to one or more other molecular entities, such as another antibody (e.g., to form a bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0089] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or different types, e.g., to create bispecific antibodies). Suitable crosslinkers include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate) crosslinkers, which have two distinct reactive groups separated by a suitable spacer. Such linkers are available, for example, from Pierce Chemical Company, Rockford, IL.

[0090] The anti-TNFα antibody, or antigen-binding portion thereof, can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups are useful to improve the biological properties of the antibody, e.g., to increase serum half-life.

[0091] The antibodies or antigen-binding portions of the present invention may also be labeled. As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into the antibody. In some embodiments, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to the polypeptide that is detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity detectable by optical or colorimetric methods). In some embodiments, the label or marker can be therapeutic, such as a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaliferase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.), gadolinium chelates, etc. Toxins such as magnetic agents, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0092] In certain embodiments, the antibodies of the present invention can exist in neutral form (including zwitterionic forms), or as positively or negatively charged species. In some embodiments, the antibodies may be complexed with counterions to form pharmaceutically acceptable salts.

[0093] bispecific binding molecules In a further aspect, the present invention provides bispecific binding molecules having the binding specificity of an anti-TNFα antibody described herein (e.g., comprising six CDRs or antigen-binding portions such as a VH and VL) and the binding specificity of a second, distinct antibody. The second antibody may be, for example, another anti-TNFα antibody (such as another antibody described herein) or may be an antibody that targets another protein, such as another cell surface molecule with activity mediating autoimmune or inflammatory disease. In certain embodiments, the second antibody targets IL17A, IL23, or angiopoietin 2.

[0094] The present invention also contemplates multispecific antibodies having the binding specificity of an anti-TNFα antibody described herein and the binding specificity of one or more additional antibodies (e.g., two or three additional antibodies).

[0095] In certain embodiments, the bispecific binding molecules described herein are used in place of the anti-TNFα antibodies or antigen-binding portions described herein in any aspect of the invention (e.g., the therapeutic methods, products, or kits described herein).

[0096] immune complex In a further aspect, the present invention provides an immunoconjugate comprising an anti-TNFα antibody or antigen-binding portion described herein conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is an anti-inflammatory agent or an immunosuppressant. In certain embodiments, the therapeutic agent is a steroid, such as a glucocorticoid receptor modulator (e.g., an agonist). For example, the therapeutic agent can be selected from dexamethasone, prednisolone, budesonide, and the like. In some embodiments, the therapeutic agent can be any payload described in PCT patent application WO 2021 / 161263 or WO 2017 / 210471, both of which are incorporated herein by reference in their entireties.

[0097] In certain embodiments, the therapeutic agent may have the structure of Formula I: [ka]

[0098] In certain embodiments, the therapeutic agent may have the structure of Formula II: [ka]

[0099] In certain embodiments, the immunoconjugates described herein are used in place of the anti-TNFα antibodies or antigen-binding portions described herein in any aspect of the invention (e.g., the therapeutic methods, products, or kits described herein).

[0100] Nucleic Acid Molecules and Vectors The present invention also provides nucleic acid molecules and sequences encoding the anti-TNFα antibodies or antigen-binding portions described herein. In certain embodiments, different nucleic acid molecules encode the heavy and light chain amino acid sequences of an anti-TNFα antibody or antigen-binding portion. In other embodiments, the same nucleic acid molecule encodes the heavy and light chain amino acid sequences of an anti-TNFα antibody or antigen-binding portion. Thus, the present invention provides isolated nucleic acid molecules comprising a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof, or a nucleotide sequence encoding the light chain or antigen-binding portion thereof, or both, of an anti-TNFα antibody or antigen-binding portion described herein.

[0101] A reference to a nucleotide sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass the complementary strand, having the complementary sequence. As used herein, the term "polynucleotide" means a polymer of nucleotides at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The term includes single- and double-stranded forms.

[0102] In any of the above embodiments, the nucleic acid molecule may be isolated. As used herein, a nucleic acid molecule referred to as "isolated" or "purified" is (1) a nucleic acid that is separated from the nucleic acid of its source, genomic DNA or cellular RNA, and / or (2) a nucleic acid that is not naturally occurring.

[0103] In some embodiments, nucleic acid molecules of the invention comprise a nucleotide sequence encoding the H-CDR1-3 and / or L-CDR1-3 of an anti-TNFα antibody or antigen-binding portion thereof. In some embodiments, nucleic acid molecules of the invention comprise a nucleotide sequence encoding the VH and / or VL of an anti-TNFα antibody or antigen-binding portion thereof. In some embodiments, nucleic acid molecules of the invention comprise a nucleotide sequence encoding the HC(s) and / or LC of an anti-TNFα antibody or antigen-binding portion thereof.

[0104] In some embodiments, the nucleic acid molecules of the invention comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73.

[0105] In certain embodiments, the nucleic acid molecule(s) of the invention comprise the following nucleotide sequence: - SEQ ID NOs: 29 and 15; - SEQ ID NOs: 31 and 15; - SEQ ID NOs: 33 and 15; SEQ ID NOs: 35 and 37; - SEQ ID NOs: 13 and 39; - SEQ ID NOs: 13 and 41; - SEQ ID NOs: 13 and 43; - SEQ ID NOs: 13 and 45; - SEQ ID NOs: 13 and 47; - SEQ ID NOs: 29 and 39; - SEQ ID NOs: 29 and 41; - SEQ ID NOs: 29 and 43; - SEQ ID NOs: 29 and 45; - SEQ ID NOs: 31 and 39; - SEQ ID NOs: 31 and 41; - SEQ ID NOs: 31 and 43; - SEQ ID NOs: 31 and 45; - SEQ ID NOs: 33 and 39; - SEQ ID NOs: 33 and 41; - SEQ ID NOs: 33 and 43; - SEQ ID NOs: 33 and 45; - SEQ ID NOs: 49 and 7; SEQ ID NOs: 51 and 7; - SEQ ID NOs: 53 and 7; - SEQ ID NOs: 5 and 55; - SEQ ID NOs: 5 and 57; - SEQ ID NOs: 5 and 59; - SEQ ID NOs: 5 and 61; - SEQ ID NOs: 5 and 63; - SEQ ID NOs: 5 and 65; - SEQ ID NOs: 5 and 67; -SEQ ID NOs: 5 and 69; SEQ ID NOs: 5 and 71; or SEQ ID NOs: 5 and 73.

[0106] In any of the above aspects of the nucleic acid molecule(s), the nucleotide sequences are on the same nucleic acid molecule or on a set of nucleic acid molecules.

[0107] The present invention further provides vectors comprising nucleic acid molecules encoding the heavy and light chains, or antigen-binding portions thereof, of the anti-TNFα antibodies described herein. In certain embodiments, the vectors of the present invention comprise the nucleic acid molecules described herein. The vectors may further comprise expression control sequences.

[0108] As used herein, the term "expression control sequence" refers to polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. In prokaryotes, such control sequences generally include a promoter, ribosomal binding site, and transcription termination sequence. In eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" is intended to include, at a minimum, all components essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0109] In some embodiments of the nucleic acid molecule(s) described herein, the nucleotide sequence may be arranged as two coding sequences (e.g., in a heterodimeric monovalent antibody described herein, comprising a first coding sequence encoding the VH, VL, CH1, and Fc monomer regions, and a second coding sequence encoding a truncated HC) or three coding sequences (e.g., in a heterotrimeric monovalent antibody described herein, a first and second coding sequence encoding the antigen binding protein HC and LC sequences, respectively, and a third coding sequence encoding an additional truncated HC). In certain embodiments, the coding sequences are present in a polycistronic arrangement on a single nucleic acid molecule. The coding sequences of a polycistronic construct can be separated from each other by, for example, a coding sequence for a self-cleaving peptide or can be separated by an internal ribosome entry site (IRES). Thus, a polycistronic construct can be transcribed as a single RNA that is processed and translated as separate polypeptides. In other embodiments, the coding sequences are on two or three separate nucleic acid molecules (e.g., in the case of heterodimeric and heterotrimeric antibodies, respectively). The coding sequences may be under the control of the same promoter or different promoters.

[0110] Host cells and antibody production methods The present invention also provides methods for producing the antibodies and antigen-binding portions thereof described herein. In some embodiments, the present invention provides host cells comprising nucleotide sequences encoding the heavy chain(s) and light chain of an anti-TNFα antibody or antigen-binding portion described herein, wherein the nucleotide sequences can be present on the same or different nucleic acid molecules. In some embodiments, the host cell comprises one or more vectors described herein. In some embodiments, the present invention provides methods for producing an anti-TNFα antibody or antigen-binding portion described herein, comprising providing the host cell; culturing the host cell under conditions suitable for expression of the antibody or antigen-binding portion; and isolating the resulting antibody or antigen-binding portion. Antibodies or antigen-binding portions produced by expression in such recombinant host cells are referred to herein as "recombinant" antibodies or antigen-binding portions. The present invention also provides progeny of such host cells, and antibodies or antigen-binding portions produced thereby.

[0111] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. By definition, a recombinant host cell does not exist in nature. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in the progeny due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0112] Nucleic acid molecules encoding the anti-TNFα antibodies and antigen-binding portions thereof described herein, as well as vectors containing these nucleic acid molecules, can be used to transfect suitable mammalian, plant, bacterial, or yeast host cells. In some embodiments, the nucleotide sequence encoding the light chain is transfected into cells at a ratio of, for example, 4:1, 2:1, or 1:1 relative to the nucleotide sequence encoding the heavy chain. In some embodiments, when the TNFα antibody has a heterotrimeric structure as described herein, the nucleotide sequences encoding the antibody light chain, the "knob" heavy chain (e.g., a truncated heavy chain), and the "hole" heavy chain (e.g., an antibody heavy chain) can be transfected at a ratio of, for example, 4:2:1 or 6:2:1. Transformation can be by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into nuclei. Additionally, nucleic acid molecules can be introduced into mammalian cells by viral vectors.

[0113] Antibodies expressed in different cell lines or transgenic animals may have different glycosylation patterns from each other. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modifications.

[0114] In some embodiments, the host cell of the invention comprises a nucleotide sequence encoding the H-CDR1-3 and / or L-CDR1-3, VH and / or VL, or HC(s) and / or LC of an anti-TNFα antibody or antigen-binding portion of the invention.

[0115] In some embodiments, the host cells of the invention comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, and 73.

[0116] In certain embodiments, the host cell of the invention comprises the following nucleotide sequence: - SEQ ID NOs: 29 and 15; - SEQ ID NOs: 31 and 15; - SEQ ID NOs: 33 and 15; SEQ ID NOs: 35 and 37; - SEQ ID NOs: 13 and 39; - SEQ ID NOs: 13 and 41; - SEQ ID NOs: 13 and 43; - SEQ ID NOs: 13 and 45; - SEQ ID NOs: 13 and 47; - SEQ ID NOs: 29 and 39; - SEQ ID NOs: 29 and 41; - SEQ ID NOs: 29 and 43; - SEQ ID NOs: 29 and 45; - SEQ ID NOs: 31 and 39; - SEQ ID NOs: 31 and 41; - SEQ ID NOs: 31 and 43; - SEQ ID NOs: 31 and 45; - SEQ ID NOs: 33 and 39; - SEQ ID NOs: 33 and 41; - SEQ ID NOs: 33 and 43; - SEQ ID NOs: 33 and 45; - SEQ ID NOs: 49 and 7; SEQ ID NOs: 51 and 7; - SEQ ID NOs: 53 and 7; - SEQ ID NOs: 5 and 55; - SEQ ID NOs: 5 and 57; - SEQ ID NOs: 5 and 59; - SEQ ID NOs: 5 and 61; - SEQ ID NOs: 5 and 63; - SEQ ID NOs: 5 and 65; -SEQ ID NOs: 5 and 67; -SEQ ID NOs: 5 and 69; SEQ ID NOs: 5 and 71; or SEQ ID NOs: 5 and 73.

[0117] Pharmaceutical Composition Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient (or as the only active ingredient) an anti-TNFα antibody or antigen-binding portion thereof, bispecific binding molecule, or immunoconjugate of the invention. In some embodiments, the pharmaceutical composition is intended for the amelioration, prevention, and / or treatment of an autoimmune or inflammatory disease, such as the conditions described herein.

[0118] Generally, the antibodies and antigen-binding portions, bispecific binding molecules, and immunoconjugates of the invention are suitable for administration as a formulation in association with one or more pharmaceutically acceptable excipient(s), e.g., as described below.

[0119] The term "excipient" is used herein to refer to any component other than the compound(s) of the present invention. The choice of excipient largely depends on factors such as the particular mode of administration, the excipient's effect on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, absorption delaying agents, and the like. Examples of pharmaceutically acceptable excipients include water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferable to include isotonicity agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Examples of pharmaceutically acceptable substances include minor amounts of wetting agents or auxiliary substances that enhance the shelf life or effectiveness of antibodies, such as wetting agents, emulsifying agents, preservatives, buffers, etc.

[0120] The pharmaceutical composition of the present invention and its manufacturing method can be easily understood by those skilled in the art. Such compositions and their manufacturing methods are described, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical composition is preferably manufactured under GMP (Good Manufacturing Practice) conditions.

[0121] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in bulk as a single unit dose or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0122] Pharmaceutical composition formulations suitable for parenteral administration (e.g., subcutaneous administration) generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In some embodiments of parenteral formulations, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations include aqueous solutions, which may contain excipients such as salts, carbohydrates, and buffers (preferably pH 3-9), although depending on the application, they may be more suitably formulated as sterile non-aqueous solutions or as dry forms for use with a suitable vehicle, such as sterile, pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol or dextrose. Such dosage forms may be suitably buffered, if necessary. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline or liposomal form.

[0123] Therapeutic Uses of the Antibodies and Compositions of the Invention In some embodiments, the anti-TNFα antibodies or antigen-binding portions, bispecific binding molecules, or immunoconjugates of the invention are used to treat a condition in a patient, such as cancer, a pulmonary condition, an intestinal condition, or a cardiac condition. In certain embodiments, the condition is an autoimmune or inflammatory condition. The patient may be a mammal, such as a human.

[0124] In some embodiments, the patient is diagnosed with arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, gouty arthritis, juvenile idiopathic arthritis (e.g., polyarticular juvenile idiopathic arthritis), spondyloarthritis (e.g., peripheral spondyloarthritis or axial spondyloarthritis), osteoarthritis, oligodendritis, erosive polyarthritis, or tendinitis-associated arthritis), Crohn's disease, ulcerative colitis, enteritis, inflammatory bowel disease, psoriasis (e.g., plaque psoriasis, pustular psoriasis, psoriasis vulgaris, pustular psoriasis, psoriasis purulentis ... Psoriasis, plaque psoriasis, nail psoriasis, etc.), ankylosing spondylitis, rheumatoid spondylitis, hidradenitis suppurativa, pyoderma gangrenosum, Netherton syndrome, Dupuytren's disease, Hermansky-Pudlak syndrome, atopic dermatitis, asthma, allergies, uveitis (e.g., panuveitis or non-infectious uveitis), age-related macular degeneration, diabetic retinopathy, scleritis, Rasmussen's encephalitis, asthma, sarcoidosis (e.g., cutaneous sarcoidosis) sis), arteritis (e.g., Takayasu's arteritis or giant cell arteritis), vasculitis, Kawasaki disease, Behçet's disease, ileal cystitis, hepatitis, nephrotic syndrome, atherosclerosis, glomerulosclerosis (e.g., focal segmental glomerulosclerosis), multiple sclerosis, mucopolysaccharidoses (e.g., type I, type II, or type IV), diabetes (e.g., type I diabetes or autoimmune diabetes), myocarditis, interstitial cystitis, inflammatory bone disorders, osteomyelitis (e.g., chronic nonbacterial osteomyelitis or chronic recurrent multifocal osteomyelitis), osteoporosis, sciatica, chronic granulomatous disease, systemic lupus erythematosus (SLE), autoimmune thyroid disorders (e.g., Hashimoto's disease), transplant rejection, graft-versus-host disease, obstructive sleep apnea, amyloidosis, neuropsychiatric disorders (e.g., neurodegenerative diseases), and neurodegenerative diseases (e.g., Alzheimer's disease). In some embodiments, the condition may be a chronic or acute condition, and / or may be an adult or pediatric condition.

[0125] In certain embodiments, the autoimmune disease (condition) or inflammatory disease (condition) is rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, ankylosing spondylitis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, hidradenitis suppurativa, polyarticular juvenile idiopathic arthritis, panuveitis, or Alzheimer's disease.

[0126] In some embodiments, the patient to be treated with an anti-TNFα antibody or antigen-binding portion, bispecific binding molecule, or immunoconjugate of the invention has received prior treatment for the condition being treated (e.g., an autoimmune disease or inflammatory disease). In other embodiments, the patient has not received such prior treatment. In some embodiments, the patient has failed prior treatment for the condition (e.g., a prior TNFα-targeted therapy).

[0127] "Treat," "treating," and "treatment" refer to a manner in which a biological disorder and / or at least one of its attendant symptoms is alleviated or eliminated. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, as used herein, "treatment" includes curative, palliative, and prophylactic treatment.

[0128] The anti-TNFα antibodies or antigen-binding portions, bispecific binding molecules, or immunoconjugates of the invention can be administered in a therapeutically effective amount to a patient having a condition described herein. By "therapeutically effective amount" is meant the amount of therapeutic agent administered that will alleviate to some extent one or more symptoms of the disorder being treated and / or produce the clinical endpoint(s) desired by a medical professional.

[0129] The anti-TNFα antibodies or antigen-binding portions, bispecific binding molecules, or immunoconjugates of the invention can be administered without additional therapeutic treatment, i.e., as monotherapy (monotherapy). Alternatively, treatment with the anti-TNFα antibodies or antigen-binding portions, bispecific binding molecules, or immunoconjugates of the invention can include at least one additional therapeutic treatment (combination therapy). In some embodiments, the anti-TNFα antibodies or antigen-binding portions, bispecific binding molecules, or immunoconjugates can be co-administered or formulated with other drugs / agents for the treatment of related conditions (e.g., autoimmune or inflammatory diseases).

[0130] In some embodiments, the anti-TNFα antibody or antigen-binding portion, bispecific binding molecule, or immunoconjugate of the invention is administered with or without methotrexate, prednisone, betamethasone, Enstilar (登録商標) , calcipotriol, metronidazole, azathioprine, tacrolimus, hydroxychloroquine, oral glucocorticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), baricitinib, ciprofloxacin, leflunomide, exenatide, teriparatide, sulfasalazine, thiopurines, 6-mercaptopurine, 2'-fucosylactose, abatacept, etanercept, infliximab, rituximab, tocilizumab, vedolizumab, golimumab, certolizumab, ustekinumab, sarilumab, andecaliximab, anakinra, NK cell lectin-like receptor subfamily K antagonists (e.g., tesunatilimab), antithymocyte globulin, IL-2, homocysteine modulating agents (e.g., vitamin B12, vitamin B6, folic acid, etc.), and radiation.

[0131] It is understood that the antibodies and antigen-binding portions thereof, bispecific binding molecules, and immunoconjugates of the present invention can be used in the methods of treatment described herein, can be used in the treatments described herein, and / or can be used in the manufacture of medicaments for the treatments described herein. It is also understood that the treatments described herein can be carried out not only using the anti-TNFα antibodies or antigen-binding portions thereof, bispecific binding molecules, or immunoconjugates thereof of the present invention, but also using any of the related pharmaceutical compositions described herein. The present invention also provides kits and articles of manufacture comprising the antibodies and antigen-binding portions thereof, bispecific binding molecules, immunoconjugates, or pharmaceutical compositions described herein.

[0132] Dosage and route of administration The antibodies or antigen-binding portions thereof, bispecific binding molecules, and immunoconjugates of the invention can be administered in an amount effective to treat the condition in question, i.e., at dosages and for periods of time necessary to achieve the desired result. A therapeutically effective amount can vary depending on factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether the antibodies, bispecific binding molecules, and immunoconjugates are administered as the sole treatment or in combination with one or more additional treatments for autoimmune and / or inflammatory diseases.

[0133] Dosage regimens can be adjusted to provide the optimum desired response. For example, a single bolus administration, several divided doses administered over time, or the dosage can be proportionally reduced or increased depending on the exigencies of the therapeutic situation. Parenteral compositions are particularly advantageously formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the patient / subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are generally dictated by and directly depend on (a) the unique characteristics of the therapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technology of compounding such active compounds for the therapeutic treatment of hypersensitivity in individuals.

[0134] Therefore, based on the disclosure provided herein, those skilled in the art can understand that dosage and administration regimen can be adjusted according to well-known methods in the therapeutic art. That is, the maximum tolerated dose can be easily set, the effective amount that produces a detectable therapeutic effect in a patient can also be determined, and the time requirement for administering each drug to produce a detectable therapeutic effect in a patient can also be determined. Therefore, although specific dosage and administration regimen are exemplified herein, these examples do not in any way limit the dosage and administration regimen that can be provided to a patient when implementing the present disclosure.

[0135] It should be noted that dosage values vary depending on the type and severity of the condition to be alleviated and may include single or multiple doses. Furthermore, it is understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the embodied compositions. Furthermore, dosage regimens for the compositions of the present invention may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, condition, severity, route of administration, and the particular antibody used. Thus, dosage regimens may vary but can be routinely determined using standard methods. For example, dosages can be adjusted based on pharmacokinetic or pharmacodynamic parameters, including clinical effects such as toxic effects and / or laboratory values. Accordingly, the present disclosure encompasses intrapatient dose escalation as determined by those skilled in the art. Determining appropriate dosages and regimens is well known in the relevant art and is understood to be within the skill of those skilled in the art given the teachings disclosed herein.

[0136] A therapeutically effective amount is measured by its ability to stabilize disease progression and / or ameliorate symptoms in a patient, preferably by its ability to reverse disease progression. The ability of an antibody, antigen-binding portion, bispecific binding molecule, immunoconjugate, or pharmaceutical composition of the invention to inhibit autoimmune or inflammatory diseases can be assessed in in vitro assays, such as those described in the Examples, as well as in appropriate animal models predictive of efficacy in humans. An appropriate dosage regimen will be selected to provide the optimal therapeutic response in each particular situation, and can be administered, for example, as a single bolus or continuous infusion, with dosage adjustments possible according to the exigencies of each case.

[0137] The antibodies or antigen-binding portions thereof, bispecific binding molecules, immunoconjugates, and pharmaceutical compositions of the present invention can be administered by any art-accepted method for administering peptides, proteins, or antibodies, and are generally suitable for parenteral administration. As used herein, "parenteral administration" includes any route of administration characterized by physically disrupting the target tissue and administering through the tissue breach, generally resulting in direct administration into the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administration by injection, application through a surgical incision, application through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration includes, but is not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular, intrasternal, intraarterial, intrathecal, intraurethral, intracranial, and intrasynovial injection or infusion. In a specific embodiment, the antibodies or antigen-binding portions thereof, bispecific binding molecules, immunoconjugates, or pharmaceutical compositions described herein are administered subcutaneously.

[0138] Diagnostic Uses and Compositions The antibodies and antigen-binding portions of the present invention are also useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the antibodies and antigen-binding portions can be used to detect and / or measure levels of TNFα in a sample from a patient (e.g., a tissue sample or a bodily fluid sample, such as inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine). Such detection can be useful, for example, in predicting whether a patient will respond to TNFα antibody therapy. Suitable detection and measurement methods include immunological methods, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistology. The present disclosure further encompasses kits (e.g., diagnostic kits) comprising the antibodies and antigen-binding portions described herein.

[0139] Products and Kits The present invention also provides articles of manufacture, e.g., kits, that include one or more containers (e.g., single-use or multi-use containers) containing a pharmaceutical composition of an anti-TNFα antibody or antigen-binding portion, bispecific binding molecule, or immunoconjugate of the invention; optionally, an additional biologically active molecule (e.g., another therapeutic agent); and instructions for use. The anti-TNFα antibody or antigen-binding portion, bispecific binding molecule, or immunoconjugate, and optionally, the additional biologically active molecule, can be packaged separately in suitable packaging, such as a non-reactive glass or plastic vial or ampoule. In certain embodiments, the vial or ampoule holds a lyophilized powder containing the anti-TNFα antibody or antigen-binding portion, bispecific binding molecule, or immunoconjugate, and / or the additional biologically active molecule. In certain embodiments, the vial or ampoule holds a concentrated stock (e.g., 2x, 5x, 10x, or more) of the anti-TNFα antibody or antigen-binding portion, bispecific binding molecule, or immunoconjugate, and / or biologically active molecule. In certain embodiments, the article of manufacture, such as a kit, includes a medical device (e.g., a syringe and needle) for administering the anti-TNFα antibody or antigen-binding portion, bispecific binding molecule, or immunoconjugate, and / or biologically active molecule; and / or a suitable diluent (e.g., sterile water, saline, etc.). The article of manufacture may further include instructions for using the anti-TNFα antibody or antigen-binding portion, bispecific binding molecule, or immunoconjugate, and optionally, additional biologically active molecule, in the methods described herein. The present disclosure also includes methods for making such articles of manufacture.

[0140] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. In the case of conflict, the present specification, including definitions, will control. Furthermore, unless the context dictates otherwise, singular terms shall include the plural and plural terms shall include the singular. Throughout this specification and embodiments, the terms "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of the stated integer or group of integers, but not the exclusion of other integers or groups of integers. Unless otherwise stated, the recitation of a list of elements herein includes the elements alone or in any combination. The recitation of an embodiment herein includes that embodiment as a single embodiment or in combination with other embodiment(s) herein. All publications, patents, patent applications, and other documents mentioned herein are incorporated by reference in their entirety. To the extent that a document incorporated by reference conflicts with the disclosure contained herein, this specification supersedes and / or is intended to supersede such conflicting material. Although a number of documents are cited herein, this citation is not an admission that any of these documents form part of the general knowledge in the art.

[0141] According to this disclosure, a backward reference in a dependent claim is intended as a shorthand for a direct and clear disclosure of each combination of claims indicated by the backward reference. Furthermore, the headings herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any way.

[0142] In order that this disclosure may be better understood, the following examples are set forth, which are illustrative and are not to be construed as limiting the scope of the invention in any way. [Example]

[0143] Example Example 1. Design and Expression of Ab1 Variants To enable the rapid identification of higher affinity Ab1 variants with pH-sensitive binding, we used a phage expression system to synthesize and express Fabs in bacteria starting from previously described higher affinity Ab1 variants. The higher affinity Ab1 variants A1 and 4.2 a-6 pH-switch variants were then expressed and characterized.

[0144] material and method Cloning of Fab variants into a phage expression system DNA encoding the heavy and light chain variable regions of all constructs was synthesized as gBlock (Integrated DNA Technologies) and cloned into a phage expression vector containing the human kappa light chain constant domain and the human G1 heavy chain constant domain 1. Additionally, the vector contained a his tag and a hemagglutinin A tag at the carboxy terminus of the heavy chain to facilitate purification and detection.

[0145] Expression and quantification of Fab in the periplasmic space of Escherichia coli Cloning was verified by expression and quantification of Fab in the periplasmic space of E. coli. Briefly, XL-0 bacteria were grown in 2X YT medium at 37°C to a density of 0.9-1.1 OD600. Isopropyl β-D-thiogalactoside was then added to the cells to a final concentration of 1 mM, and 4.0 mL of the culture was transferred to a 14 mL snap-top tube. Each tube was transfected with 4 μL of high-titer phage stock and placed in a shaker (225 rpm) at 37°C. After 1 hour, the temperature was reduced to 25°C and the cells were cultured for an additional 14-16 hours. Cells were harvested by centrifugation at 3,900 rpm for 30 minutes in an Eppendorf 5810R centrifuge (~3,200 x g). The supernatant was discarded and the cells were suspended in 0.25 mL of lysis buffer (30 mM Tris, pH 8.0, 2 mM EDTA, 20% sucrose, 2 mg / mL lysozyme, 5 U / mL Dnase I) and incubated on ice for 30 minutes. The cell suspension was transferred to a 1.5 mL tube and centrifuged at 15,000 rpm for 15 minutes in an Eppendorf 5424 mini-ultracentrifuge (~21,000 x g) to pellet the cell debris. The supernatant was carefully removed without disturbing the pellet and stored at 4°C until use.

[0146] To quantify Fab expression, 50 μL / well of 2 μg / mL sheep anti-human Fd (Southern Biotech, Product No. 2046-01) was added to a 96-well Costar-3366 plate in PBS and incubated overnight at 4°C. The plate was washed three times with PBS containing 0.05% Tween 20 (PBS-T) and 50 μL / well of sample diluent was added. Samples were diluted in 1% BSA-PBS. A standard curve was prepared by serially diluting human Fab (Rockland, Product No. 009-01015) starting at 1 μg / mL. The plate was then incubated for 1 hour at 25°C, washed three times with PBS-T, and incubated with 50 μL / well of anti-kappa HRP conjugate (Southern Biotech, Product No. 2060-05), diluted 5,000-fold in PBS-T, for 1 hour at 25°C. The plate was washed three times with PBS-T and then developed with 50 μL / well of 1-Step Ultra TMB-ELISA (ThermoFisher Scientific, product number 34028). The reaction was stopped by adding 2N H2SO4, and A450 was measured before and after the addition of H2SO4 using a Spectramax plate reader.

[0147] result The mutants that were expressed and characterized are summarized in Table 1 below. [Table 1-1] [Table 1-2]

[0148] Table 2 below lists the CDR sequences of the variants. [Table 2]

[0149] Example 2. Characterization of Ab1 Fab, Ab1 variant Fab, and Ab4 Fab by ELISA Introducing histidine residues into the CDRs often reduces binding at pH 7.4. Consequently, to generate Ab1 pH-switch variants with higher affinity at pH 7.4, we used previously identified higher-affinity Ab1 variants (Table 1: A1, cb1-3, 4.2 a-6) as starting points (templates) for introducing histidine residues into the CDRs. Prior to generating the pH-switch variants, the relative binding of Ab1 Fab and Ab1 variant Fab was assessed by ELISA. Furthermore, we characterized the relative binding of Ab4 Fab, which has been reported to bind TNFα with higher affinity than Ab1.

[0150] material and method ELISA characterization of Fab binding to soluble biotinylated TNFα with extensive washing in the presence of soluble TNFα A 96-well Costar-3366 plate was treated with 50 μL / well of 2 μg / mL sheep anti-human Fd (Southern Biotech, product number 2046-01) in PBS for 1 hour at room temperature. The plate was washed four times with PBS containing 0.05% Tween 20 (PBS-T) and blocked with 100 μL / well of 1% BSA-PBS for 1 hour at room temperature. The block was removed, and 50 μL / well of 1 μg / mL sample was added. Sample dilutions were performed in 1% BSA-PBS. After washing the plate four times with PBS containing 0.05% Tween 20 (PBS-T), biotinylated human TNFα was serially diluted 3-fold starting from 60 nM in B-PBS (50 μL / well) and incubated for 1 hour at 25°C. Plates were washed four times with PBS-T, and 50 μL / well of 100 nM human TNFα in 1% BSA was added and incubated at 25°C for 20 hours. Plates were washed four times with PBS-T and incubated with 50 μL / well of Neutravidin HRP (ThermoFisher Scientific, Catalog No. 31030) for 1 hour at 25°C. After washing four times with PBS-T, plates were developed with 50 μL / well of One-Step Ultra TMB-ELISA (ThermoFisher Scientific, Product No. 34028). The reaction was stopped by the addition of 2N H2SO4, and the A450 was measured before and after the addition of H2SO4 using a Spectramax plate reader.

[0151] result Characterization of the variants using standard ELISA conditions with multiple rapid washes did not allow us to distinguish the binding affinities of the Fab variants from those of Ab1 Fab (data not shown). However, binding of soluble biotinylated TNFα to immobilized Fab followed by a prolonged dissociation step (20 h) in the presence of 100 nM non-biotinylated TNFα allowed us to distinguish differences in binding affinity. Under these conditions, Ab1 Fab bound weakest, as evidenced by the lowest maximum binding signal (Figure 1). The Ab4 Fab variants and Ab1 Fab variants all bound TNFα with higher affinity than Ab1 Fab (Figure 1). Ab4 Fab bound more tightly than Ab1 Fab, but weaker than the three Ab1 Fab variants with increased affinity, A1, cb1-3, and 4.2a-6 (Figure 1). As a result, the variable regions of A1, cb1-3, and 4.2 a-6 can all serve as templates for generating higher affinity Ab1 pH-switch variants.

[0152] Example 3. Characterization of pH sensitivity of binding of Ab1, Ab1 variants, Ab4, AF-M2631 Fab, and AF-M2637 Fab Next, the pH sensitivity of binding of the higher affinity Ab1 Fab variants was compared with that of Ab1, Ab4, and the previously identified pH-switch variants of Ab1, AF-M2631 and AF-M2637. To identify the relative binding avidity of TNFα Fabs at both pH 7.4 and pH 6.0, a modified ELISA was used. Briefly, Fab binding to immobilized TNFα□ was followed by a prolonged dissociation step (2 h) in the presence of 100 nM TNFα at either pH 7.4 or pH 6.0.

[0153] material and method ELISA characterization of Fab binding to immobilized TNFα in the presence of soluble TNFα with extensive washing. A 96-well Costar-3366 plate was coated with 50 μL / well of 1 μg / mL human TNFα (Genscript catalog number Z01001) in PBS for 1 hour at room temperature. The plate was washed twice with PBS-T and blocked with 100 μL / well of 1% BSA in PBS (B-PBS) for 1 hour at 25°C. Fab samples were serially diluted 3-fold starting at 40 nM in B-PBS and incubated for 1 hour at 25°C (50 μL / well). The plate was washed four times with PBS-T, and 50 μL / well of 100 nM human TNFα in 1% BSA was added and incubated for 2 hours at 25°C. The plate was washed four times with PBS-T, and 50 μL / well of anti-human kappa, HRP conjugate (Southern Biotech, product number 2060-05) diluted 1:5000 in B-PBS was added for 1 hour at 25°C. Plates were washed three times with PBS-T and then developed with 50 μL / well of 1-Step Ultra TMB-ELISA (ThermoFisher Scientific, product number 34028). The reaction was stopped by adding 2N H2SO4, and the A450 was measured before and after the addition of H2SO4 using a Spectramax plate reader.

[0154] result Characterization of Fab binding to immobilized TNFα at pH 7.4 showed that Ab1, A1, 4.2a-6, and Ab4 all bound with similar high affinity (Figure 2A, open symbols). The Ab1 pH-switch variant Fabs AF-M2631 and AF-M2637 bound with lower affinity, with AF-M2631 binding more strongly than AF-M2637 (Figure 2A, filled circles and filled squares, respectively). After a prolonged dissociation step at pH 6.0, binding of the pH-switch variant Fabs AF-M2631 and AF-M2637 was significantly reduced compared to the other variants (Figure 2B, compare filled symbols vs. open symbols). Additionally, Ab1 Fab appeared to exhibit pH-sensitive binding to a greater extent than that observed with the higher affinity Ab1 Fab variants A1 and 4.2a-6 or Ab4 Fab (Figure 2B, compare open circles vs. other open symbols). These data indicate that the variable regions of A1 and 4.2a-6 Fab can serve as templates for designing pH-switch variants that bind more tightly than Ab1-based pH-switch variants at pH 7.4.

[0155] Expression and Characterization of Heavy or Light Chain pH-Switch Variant Fabs Templated from Example 4.4.2a-6 Next, we expressed heavy and light chain variants of 4.2a-6 with specific histidine mutations in the CDRs as Fabs in bacteria and characterized their binding after extended dissociation at pH 7.4 or pH 6.0. Ab1, AF-M2631, AF-M2637, and eight 4.2a-6 variants designated 4.2a-6-VH1, 4.2a-6-VH2, 4.2a-6-VH3, 4.2a-6-H, 4.2a-6-VL1, 4.2a-6-VL2, 4.2a-6-VL4, and 4.2a-6-VL5 (Table 1) were analyzed. The ELISA method used to characterize these variants is described in Example 3.

[0156] result All histidine heavy or light chain CDR Fab variants of the 4.2a-6 template bound TNFα better than AF-M2631 Fab after a prolonged dissociation step at pH 7.4 (Figure 3A, compare filled symbols with open triangles). Most variants showed similar, albeit slightly weaker, binding avidity to Ab1 Fab (Figure 3A, compare filled symbols with open circles). Fab variant 4.2a-6-VH2 showed stronger binding than Ab1 Fab (Figure 3A, compare filled squares with open circles), whereas Fab variant 4.2a-6-VL1 showed very similar binding to Ab1 Fab (Figure 3A, compare filled inverted triangles with open circles). Most of the Fab variants bound similarly to Ab1 Fab after a prolonged dissociation step at pH 6.0 (Figure 3B, compare filled symbols with open circles). However, Fab variant 4.2a-6-VL5 showed reduced binding compared to AF-M2631 Fab (Figure 3B, compare filled star and open triangle). Fab variant 4.2a-6-VL5 exhibited strong pH-dependent binding, with strong binding at pH 7.4 and significantly reduced binding at pH 6.0. Based on sequence homology, Fab variant 4.2a-6-VL7 is expected to exhibit properties similar to Fab variant 4.2a-6-VL5.

[0157] Expression and Characterization of Heavy and Light Chain Combinatorial pH-Switch Variant Fabs Templated from Example 5.4.2a-6 Individual 4.2a-6 heavy and light chain pH-switch variant sequences were combined (Table 1, 4.2a-6 template combinatorial pH-switch variants) to determine whether stronger pH sensitivity at pH 6.0 could be identified. The ELISA method used to characterize these variants is described in Example 3.

[0158] result All combinatorial Fab variants containing the 4.2a-6-VH1 heavy chain exhibited significantly weaker binding than the AF-M2637 Fab after prolonged dissociation at pH 7.4 (Figure 4A, compare filled and open symbols). Furthermore, the variants did not show a significant decrease in binding after prolonged dissociation at pH 6.0, indicating that combinatorial histidine mutations containing 4.2a-6-VH1 are not effective in creating a pH switch on the 4.2a-6 template (Figure 4B). Similar results were observed with combinatorial Fab variants containing the 4.2a-6-VH2 heavy chain. Specifically, 4.2a-6-VH2xVL1 and 4.2a-6-VH2xVL2 exhibited weak binding after prolonged dissociation at pH 7.4 (Figure 4A) and little pH switch activity at pH 6.0 (Figure 4B). The 4.2a-6-VH2xVL4 variant showed little binding after prolonged dissociation at both pH 7.4 (Figure 4C) and pH 6.0 (Figure 4D). The 4.2a-6-VH2xVL5 variant showed greater binding than AF-M2637 but less than AF-M2631 after prolonged dissociation at pH 7.4 (Figure 4C). However, 4.2a-6-VH2xVL5 did not exhibit a pH switch upon dissociation at pH 6.0 (Figure 4D). All combinatorial Fab variants containing the 4.2a-6-VH3 heavy chain showed significantly weaker binding than AF-M2637 after prolonged dissociation at pH 7.4 (Figure 4C, compare filled and open symbols). Furthermore, after prolonged dissociation at pH 6.0, the mutants did not show a significant decrease in binding, indicating that the combinatorial histidine mutations comprising 4.2a-6-VH3 were not effective in creating a pH switch on the 4.2a-6 template (Figure 4D). Taken together, these data indicate that the combinatorial pH switch constructed in this study based on the 4.2a-6 template is not useful in a monovalent form.

[0159] Example 6. Expression and characterization of heavy or light chain pH-switch variant Fabs using A1 as a template Next, heavy and light chain variants of A1 containing specific histidine mutations in the CDRs were expressed as Fabs in bacteria and characterized for binding after extended dissociation at pH 7.4 or pH 6.0. The Ab1, AF-M2631, and AF-M2637 variants, as well as 13 A1 variants designated A1-VH1, A1-VH2, A1-VH3, A1-VL1, A1-VL2, A1-VL3, A1-VL4, A1-VL5, A1-VL-6, A1-VL7, A1 LC Q27H, A1 LC G28H, and A1 LC I29 H, were analyzed (Table 1). The ELISA method used to characterize these variants is described in Example 3.

[0160] result All Fab variants except A1-VL5 and A1-VL7 bound more strongly than AF-M2631 Fab after prolonged dissociation at pH 7.4 (Figures 5A and 5C). A1-VL5 and A1-VL7 showed similar binding to AF-M2631 Fab (Figure 5C, compare filled triangles and filled circles with open triangles). Fab variants A1-VH1 (Figure 5B, filled circles), A1-VH3 (Figure 5B, filled triangles), A1-VL1 (Figure 5B, inverted filled triangles), A1-VL5 (Figure 5D, filled circles), and A1-VL7 (Figure 5D, filled triangles) all showed enhanced dissociation at pH 6.0 relative to pH 7.4. Consequently, all of these monovalent Fab variants may be useful as pH-sensitive modulators of TNFα binding and neutralization. In contrast, A1 LC Q27H, A1 LC G28H, and A1 LC I29H bound more tightly than Ab1 Fab after prolonged dissociation at pH 6.0, and therefore these three Fab variants are not useful as pH switches.

[0161] Example 7. Characterization of certain A1 template heavy or light chain pH-switch Fab variants Specific Fab variants identified in Example 6 were recharacterized and compared to the Fabs of Ab1, AF-M2631, AF-M2637, and A1. The ELISA methods used to characterize these variants are described in Example 3.

[0162] result All Fab variants showed stronger binding than AF-M2637 Fab after prolonged dissociation at pH 7.4 (Figure 6A, compare filled symbols and open squares). Mutants A1-VH1 (filled circles) and A1-VH3 (filled squares) bound more strongly than AF-M2631 Fab (open triangles), while A1-VL1 (filled triangles), A1-VL5 (filled inverted triangles), and A1-VL7 (filled diamonds) showed binding similar to AF-M2631 Fab. Binding of template A1 Fab (filled inverted triangles) was indistinguishable from Ab1 Fab (open circles) under these assay conditions. All variants showed accelerated dissociation at pH 6.0 (Figure 6B, filled symbols). A1-VH1 (closed circles) and A1-VH3 (closed squares) showed slightly stronger binding than AF-M2631 (open triangles) at pH 6.0 dissociation, while A1-VL1 (closed triangles) and A1-VL5 (closed inverted triangles) showed weaker binding than AF-M2631 at pH 6.0 dissociation. A1-VL7 showed the weakest binding at pH 6.0 dissociation, comparable to AF-M2637 (open squares). All variants showed tight pH-dependent binding in the monovalent Fab format, with strong binding at pH 7.4 and significantly reduced binding at pH 6.0.

[0163] Example 8. Characterization of Fab variants binding to mouse TNFα The effect of reduced binding of the mutants at pH 6.0 on immunogenicity can be screened using wild-type mice. However, before conducting in vivo studies, we characterized the binding of the mutants to mouse TNFα by ELISA to determine whether the pH-switch activity observed with human TNFα was maintained with mouse TNFα.

[0164] material and method ELISA characterization of Fab binding to soluble biotinylated mouse TNF.ALPHA. with extensive washing in the presence of soluble TNF.ALPHA. A 96-well Costar-3366 plate was treated with 50 μL / well of 2 μg / mL sheep anti-human Fd (Southern Biotech, product number 2046-01) in PBS for 1 hour at room temperature. The plate was washed four times with PBS containing 0.05% Tween 20 (PBS-T) and blocked with 100 μL / well of 1% BSA-PBS for 1 hour at room temperature. The blocker was removed, and 50 μL / well of 0.5 μg / mL sample was added. Sample dilutions were performed in 1% BSA-PBS. After washing the plate four times with PBS containing 0.05% Tween 20 (PBS-T), biotinylated mouse TNFα (Acro Biosystems) was serially diluted 3-fold starting from 30 nM in B-PBS and incubated at 25°C for 1 hour (50 μL / well). The plate was washed with 500 mL of PBS-T, pH 6.0, or PBS-T, pH 7.4, for 1 hour. During this wash, the PBS-T was removed from the plate every 10 minutes. The plate was then washed four times with PBS-T and incubated with 50 μL / well of NeutrAvidin HRP (ThermoFisher Scientific, Catalog No. 31030) diluted 1:2,000 in B-PBS at 25°C for 1 hour. After washing the plate four times with PBS-T, the plate was developed with 50 μL / well of 1-Step Ultra TMB-ELISA (ThermoFisher Scientific, Product No. 34028). The reaction was stopped by adding 2N H2SO4, and the A450 was measured before and after the addition of H2SO4 using a Spectramax plate reader.

[0165] result All A1-templated pH-switches identified in Example 7 and the 4.a-6-templated pH-switch, 4.2a-6-VL5, identified in Example 4 bound mouse TNFα at pH 7.4 (Figure 7A). A1 (closed circles) and 4.2a-6 (open squares) bound tightly to mouse TNFα, whereas Ab4 and the control AF-M2637 did not bind to mouse TNFα under the conditions used in this assay (asterisks and open triangles, respectively). Ab1, the control AF-M2631, and all other variants exhibited similar intermediate binding activity.

[0166] Binding of A1 (closed circles) and 4.2a-6 (open squares) to mouse TNFα after dissociation at pH 6.0 remained strong, indicating that binding was not pH-sensitive (Figure 7B). Similarly, binding of Ab1 (open circles) was only slightly reduced after dissociation at pH 6.0. Binding of all variants except AF-M2631 (closed squares), A1-VH1 (closed triangles), and A1-VH3 (open inverted triangles) was significantly reduced after dissociation at pH 6.0. Consequently, while all variants are suitable for testing in mice, certain variants (AF-M2631, A1-VH1, and A1-VH3) may not have optimal pH sensitivity.

Claims

1. a) A heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 8; b) VH containing the amino acid sequence of SEQ ID NO: 10 and VL containing the amino acid sequence of SEQ ID NO: 12; or c) VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 16 An anti-TNFα antibody or its antigen-binding moiety that binds to the same human TNFα epitope as the reference antibody containing, Here, the anti-TNFα antibody or antigen-binding moiety comprises VH and VL, respectively, which are at least 90% identical to the VH and VL of the reference antibody; and has a lower binding affinity to TNFα at pH 6.0 than at pH 7.4; and is monovalent if required. Anti-TNFα antibody or antigen-binding moiety.

2. The antibody, a) A monovalent antigen-binding protein comprising a heavy chain (HC) containing VH which is at least 90% identical to VH of a reference antibody, and a light chain (LC) containing VL which is at least 90% identical to VL of a reference antibody; and b) Leaving the variable domain and CH1 domain, cleavage type HC This includes, where the antigen-binding protein HC and the cleaved form HC can be dimerized. The anti-TNFα antibody or antigen-binding moiety according to claim 1.

3. The anti-TNFα antibody or antigen-binding moiety according to claim 2, wherein the antigen-binding protein HC and the cleaved HC comprise knob-in-hole modifications, and optionally, the antigen-binding protein HC is isotype subclass IgG1 and comprises mutations T366S, L368A and Y407A in its CH3 domain, and the cleaved HC is isotype subclass IgG1 and comprises mutation T366W in its CH3 domain, where the residues are numbered according to the Eu system.

4. The anti-TNFα antibody or antigen-binding moiety according to claim 2 or 3, wherein the antigen-binding protein HC is isotype subclass IgG1 and comprises mutation Y349C, and the cleaved HC is isotype subclass IgG1 and comprises mutation S354C, where the residues are numbered according to the Eu system.

5. a) Sequence IDs 87, 76, 97, 88, 89, and 90, respectively; b) Sequence IDs 87, 76, 98, 88, 89, and 90, respectively; c) Sequence IDs 87, 76, 101, 88, 89, and 90, respectively; d) Sequence IDs 87, 76, 102, 88, 89, and 103, respectively; e) Sequence IDs 87, 76, 77, 104, 89, and 90, respectively; f) Sequence IDs 87, 76, 77, 88, 89 and 105, respectively; g) Sequence IDs 87, 76, 77, 88, 89 and 106, respectively; h) Sequence IDs 87, 76, 77, 107, 89, and 90, respectively; i) Sequence IDs 87, 76, 77, 104, 89, and 108, respectively; j) Sequence IDs 87, 76, 97, 104, 89, and 90, respectively; k) Sequence IDs 87, 76, 97, 88, 89 and 105, respectively; l) Sequence numbers 87, 76, 97, 88, 89 and 106, respectively; m) Sequence IDs 87, 76, 97, 107, 89, and 90, respectively; n) Sequence IDs 87, 76, 98, 104, 89, and 90, respectively; o) Sequence IDs 87, 76, 98, 88, 89, and 105, respectively; p) Sequence IDs 87, 76, 98, 88, 89, and 106, respectively; q) Sequence numbers 87, 76, 98, 107, 89, and 90, respectively; r) Sequence IDs 87, 76, 101, 104, 89, and 90, respectively; s) Sequence IDs 87, 76, 101, 88, 89, and 105, respectively; t) Sequence numbers 87, 76, 101, 88, 89 and 106 respectively; or u) Sequence numbers 87, 76, 101, 107, 89 and 90 respectively An anti-TNFα antibody or its antigen-binding moiety comprising heavy chain (HC)CDR1-3 and light chain (LC)CDR1-3.

6. The antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are a) Sequence IDs 30 and 16, respectively; b) Sequence IDs 32 and 16, respectively; c) Sequence IDs 34 and 16, respectively; d) Sequence IDs 36 and 38, respectively; e) Sequence IDs 14 and 40, respectively; f) Sequence IDs 14 and 42, respectively; g) Sequence IDs 14 and 44, respectively; h) Sequence IDs 14 and 46, respectively; i) Sequence IDs 14 and 48, respectively; j) Sequence IDs 30 and 40, respectively; k) Sequence IDs 30 and 42, respectively; l) Sequence IDs 30 and 44, respectively; m) Sequence IDs 30 and 46, respectively; n) Sequence IDs 32 and 40, respectively; o) Sequence IDs 32 and 42, respectively; p) Sequence IDs 32 and 44, respectively; q) Sequence IDs 32 and 46, respectively; r) Sequence IDs 34 and 40, respectively; s) Sequence IDs 34 and 42, respectively; t) Sequence IDs 34 and 44 respectively; or u) Sequence IDs 34 and 46, respectively The anti-TNFα antibody or antigen-binding moiety according to claim 5, comprising:

7. a) Sequence IDs 81, 76, 109, 83, 79, and 80, respectively; b) Sequence IDs 81, 76, 110, 83, 79, and 80, respectively; c) Sequence IDs 81, 76, 111, 83, 79, and 80, respectively; d) Sequence IDs 81, 76, 82, 112, 79, and 80, respectively; e) Sequence numbers 81, 76, 82, 83, 79 and 99, respectively; f) Sequence numbers 81, 76, 82, 83, 79 and 113, respectively; g) Sequence IDs 81, 76, 82, 83, 79 and 100, respectively; h) Sequence IDs 81, 76, 82, 114, 79, and 80, respectively; i) Sequence IDs 81, 76, 82, 83, 79 and 115, respectively; j) Sequence numbers 81, 76, 82, 112, 79, and 116, respectively; k) Sequence IDs 81, 76, 82, 117, 79, and 80, respectively; l) Sequence numbers 81, 76, 82, 118, 79 and 80, respectively; or m) Sequence numbers 81, 76, 82, 119, 79 and 80 respectively An anti-TNFα antibody or its antigen-binding moiety comprising heavy chain (HC)CDR1-3 and light chain (LC)CDR1-3.

8. The antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are a) Sequence IDs 50 and 8, respectively; b) Sequence IDs 52 and 8, respectively; c) Sequence IDs 54 and 8, respectively; d) Sequence IDs 6 and 56, respectively; e) Sequence IDs 6 and 58, respectively; f) Sequence numbers 6 and 60, respectively; g) Sequence IDs 6 and 62, respectively; h) Sequence IDs 6 and 64, respectively; i) Sequence numbers 6 and 66, respectively; j) Sequence IDs 6 and 68, respectively; k) Sequence IDs 6 and 70, respectively; l) Sequence IDs 6 and 72, respectively; or m) Sequence IDs 6 and 74, respectively The anti-TNFα antibody or antigen-binding moiety according to claim 7, comprising:

9. The anti-TNFα antibody or antigen-binding moiety according to any one of claims 5 to 8, wherein the antibody or antigen-binding moiety is monovalent.

10. The antibody is monovalent, and a) a monovalent antigen-binding protein comprising an HC containing the VH and an LC containing the VL; and b) Leaving the variable domain and CH1 domain, cleavage type HC The anti-TNFα antibody or antigen-binding moiety according to claim 6 or 8, comprising, wherein the antigen-binding protein HC and the cleaved form HC can be dimerized.

11. a) A monovalent antigen-binding protein comprising HC containing VH having the amino acid sequence of SEQ ID NO: 50 and LC containing VL having the amino acid sequence of SEQ ID NO: 8; and b) Leaving the variable domain and CH1 domain, cleavage type HC A monovalent anti-TNFα antibody comprising, wherein the antigen-binding protein HC and the cleaved form HC can be dimerized.

12. a) A monovalent antigen-binding protein comprising HC containing VH having the amino acid sequence of SEQ ID NO: 54 and LC containing VL having the amino acid sequence of SEQ ID NO: 8; and b) Leaving the variable domain and CH1 domain, cleavage type HC A monovalent anti-TNFα antibody comprising, wherein the antigen-binding protein HC and the cleaved form HC can be dimerized.

13. a) A monovalent antigen-binding protein comprising HC containing VH having the amino acid sequence of SEQ ID NO: 6 and LC containing VL having the amino acid sequence of SEQ ID NO: 56; and b) Leaving the variable domain and CH1 domain, cleavage type HC A monovalent anti-TNFα antibody comprising, wherein the antigen-binding protein HC and the cleaved form HC can be dimerized.

14. a) A monovalent antigen-binding protein comprising HC containing VH having the amino acid sequence of SEQ ID NO: 6 and LC containing VL having the amino acid sequence of SEQ ID NO: 64; and b) Leaving the variable domain and CH1 domain, cleavage type HC A monovalent anti-TNFα antibody comprising, wherein the antigen-binding protein HC and the cleaved form HC can be dimerized.

15. a) A monovalent antigen-binding protein comprising HC containing VH having the amino acid sequence of SEQ ID NO: 6 and LC containing VL having the amino acid sequence of SEQ ID NO: 68; and b) Leaving the variable domain and CH1 domain, cleavage type HC A monovalent anti-TNFα antibody comprising, wherein the antigen-binding protein HC and the cleaved form HC can be dimerized.

16. a) A monovalent antigen-binding protein comprising HC containing VH having the amino acid sequence of SEQ ID NO: 14 and LC containing VL having the amino acid sequence of SEQ ID NO: 44; and b) Leaving the variable domain and CH1 domain, cleavage type HC A monovalent anti-TNFα antibody comprising, wherein the antigen-binding protein HC and the cleaved form HC can be dimerized.

17. A monovalent anti-TNFα antibody according to any one of claims 11 to 16, wherein the antigen-binding protein HC and the cleaved HC comprise knob-in-hole modifications, and optionally, the antigen-binding protein HC is isotype subclass IgG1 and comprises mutations T366S, L368A and Y407A in its CH3 domain, and the cleaved HC is isotype subclass IgG1 and comprises mutation T366W in its CH3 domain, where the residues are numbered according to the Eu system.

18. The monovalent anti-TNFα antibody according to claim 17, wherein the antigen-binding protein HC is isotype subclass IgG1 and contains mutation Y349C, and the cleaved HC is isotype subclass IgG1 and contains mutation S354C, where the residues are numbered according to the Eu system.

19. The antibody is monovalent, and a) A single-stranded variable fragment (scFv) comprising the VH and the VL linked to the Fc monomer; and b) Leaving the variable domain and CH1 domain, cleavage type HC The anti-TNFα antibody according to claim 6 or 8, comprising, wherein the Fc monomer linked to the scFv and the cleaved HC can be dimerized.

20. The monovalent anti-TNFα antibody according to claim 19, wherein the Fc monomer linked to the scFv and the cleaved HC include knob-in-hole modifications, and optionally the Fc monomer linked to the scFv is isotype subclass IgG1 and includes mutants T366S, L368A and Y407A in the CH3 domain, and the cleaved HC is isotype subclass IgG1 and includes mutant T366W in the CH3 domain, where the residues are numbered according to the Eu system.

21. The monovalent anti-TNFα antibody according to claim 20, wherein the Fc monomer linked to scFv is isotype subclass IgG1 and comprises mutation Y349C, and the cleaved HC is isotype subclass IgG1 and comprises mutation S354C, where the residues are numbered according to the Eu system.

22. The anti-TNFα antibody or antigen-binding moiety according to any one of claims 5 to 8 or 11 to 16, wherein the antibody or antigen-binding moiety has a lower binding affinity to human TNFα at pH 6.0 than at pH 7.

4.

23. An anti-TNFα antibody or antigen-binding moiety according to any one of claims 1 to 3, 5 to 8, or 11 to 16, wherein the antibody or antigen-binding moiety is compared to an antibody comprising the VH amino acid sequence and VL amino acid sequence of SEQ ID NOs: 2 and 4, respectively; SEQ ID NOs: 6 and 8, respectively; SEQ ID NOs: 10 and 12, respectively; or SEQ ID NOs: 14 and 16, respectively. a) Low degradation in vivo; b) Increased reuse on the cell surface in vivo; c) It has a long half-life in vivo; d) Low immunogenicity in vivo; or e) has any combination of a) to d), In short, the antibody or antigen-binding portion does not form a large immune complex. Anti-TNFα antibody or antigen-binding moiety.

24. A bispecific binding molecule having the binding specificity of an anti-TNFα antibody according to any one of claims 1 to 3, 5 to 8, or 11 to 16, and the binding specificity of a second distinct antibody.

25. The bispecificity conjugation molecule according to claim 24, wherein the second antibody is an anti-IL17A antibody, an anti-IL23 antibody, or an anti-angiopoietin 2 (Ang2) antibody.

26. An immune complex comprising an anti-TNFα antibody or antigen-binding moiety according to any one of claims 1 to 3, 5 to 8, or 11 to 16, linked to a therapeutic agent.

27. The immune complex according to claim 26, wherein the therapeutic agent is an anti-inflammatory agent or an immunosuppressant, and if necessary, the therapeutic agent is a steroid.

28. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain and light chain sequences of an anti-TNFα antibody or antigen-binding moiety according to any one of claims 1 to 3, 5 to 8, or 11 to 16.

29. a) Sequence IDs 29 and 15; b) Sequence IDs 31 and 15; c) Sequence IDs 33 and 15; d) Sequence IDs 35 and 37; e) Sequence IDs 13 and 39; f) Sequence IDs 13 and 41; g) Sequence IDs 13 and 43; h) Sequence IDs 13 and 45; i) Sequence IDs 13 and 47; j) Sequence IDs 29 and 39; k) Sequence IDs 29 and 41; l) Sequence IDs 29 and 43; m) Sequence IDs 29 and 45; n) Sequence IDs 31 and 39; o) Sequence IDs 31 and 41; p) Sequence IDs 31 and 43; q) Sequence IDs 31 and 45; r) Sequence IDs 33 and 39; s) Sequence IDs 33 and 41; t) Sequence IDs 33 and 43; u) Sequence IDs 33 and 45; v) Sequence IDs 49 and 7; w) Sequence IDs 51 and 7; x) Sequence IDs 53 and 7; y) Sequence IDs 5 and 55; z) Sequence IDs 5 and 57; aa) Sequence IDs 5 and 59; bb) Sequence IDs 5 and 61; cc) Sequence IDs 5 and 63; dd) Sequence IDs 5 and 65; ee) Sequence IDs 5 and 67; ff) Sequence IDs 5 and 69; gg) Sequence IDs 5 and 71; or hh) Sequence IDs 5 and 73 An isolated nucleic acid molecule according to claim 28, comprising the nucleotide sequence.

30. A vector comprising an isolated nucleic acid molecule according to claim 28, the vector further comprising an expression regulatory sequence operably linked to the isolated nucleic acid molecule.

31. A host cell comprising a nucleotide sequence encoding a heavy chain sequence and a nucleotide sequence encoding a light chain sequence of an anti-TNFα antibody or antigen-binding moiety according to any one of claims 1 to 3, 5 to 8, or 11 to 16.

32. The host cell according to claim 31, wherein the host cell contains the isolated nucleic acid molecule described in claim 29.

33. A method for producing an anti-TNFα antibody or its antigen-binding portion, comprising providing the host cells described in claim 31, culturing the host cells under conditions suitable for the expression of the antibody or antigen-binding portion, and isolating the obtained antibody or antigen-binding portion.

34. A pharmaceutical composition comprising an anti-TNFα antibody or antigen-binding moiety according to any one of claims 1 to 3, 5 to 8, or 11 to 16, and a pharmaceutically acceptable excipient.

35. A pharmaceutical composition comprising an anti-TNFα antibody or antigen-binding moiety according to any one of claims 1 to 3, 5 to 8, or 11 to 16, for treating an autoimmune disease or inflammatory disease in a patient.

36. The pharmaceutical composition according to claim 35, wherein the autoimmune disease or inflammatory disease is rheumatoid arthritis, psoriatic arthritis, psoriasis vulgaris, ankylosing spondylitis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, hidradenitis suppurativa, polyarticular juvenile idiopathic arthritis, panuveitis, or Alzheimer's disease.

37. The pharmaceutical composition according to claim 35, wherein the patient should be treated with an additional therapeutic agent, the additional therapeutic agent being an anti-inflammatory agent or an immunosuppressant, and if required, the additional therapeutic agent being methotrexate.

38. A pharmaceutical composition comprising the bispecific binding molecule according to claim 24 for treating an autoimmune disease or inflammatory disease in a patient.

39. The pharmaceutical composition according to claim 38, wherein the autoimmune disease or inflammatory disease is rheumatoid arthritis, psoriatic arthritis, psoriasis vulgaris, ankylosing spondylitis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, hidradenitis suppurativa, polyarticular juvenile idiopathic arthritis, panuveitis, or Alzheimer's disease.

40. The pharmaceutical composition according to claim 38, wherein the patient is to be treated with an additional therapeutic agent, wherein the additional therapeutic agent is an anti-inflammatory agent or an immunosuppressant, and if required, the additional therapeutic agent is methotrexate.

41. A pharmaceutical composition comprising the immune complex according to claim 26 for treating an autoimmune disease or inflammatory disease in a patient.

42. The pharmaceutical composition according to claim 41, wherein the autoimmune disease or inflammatory disease is rheumatoid arthritis, psoriatic arthritis, psoriasis vulgaris, ankylosing spondylitis, axial spondyloarthritis, Crohn's disease, ulcerative colitis, hidradenitis suppurativa, polyarticular juvenile idiopathic arthritis, panuveitis, or Alzheimer's disease.

43. The pharmaceutical composition according to claim 41, wherein the patient is to be treated with an additional therapeutic agent, wherein the additional therapeutic agent is an anti-inflammatory agent or an immunosuppressant, and if required, the additional therapeutic agent is methotrexate.

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