Antibody variants

Abstract

To provide an antibody that has excellent effector function and / or pharmacokinetic properties, binds to TNFα, and shows modified FcRn bond.SOLUTION: An antibody including a TNFα binding domain and an FcRn-binding site has high affinity to human FcRn characterized in less than 500 nM of dissociation equilibrium constant (KD) at pH6, and furthermore has no affinity or low affinity to human FcRn characterized in KD larger than 10 μM at pH7.4, where an amino acid sequence of the antibody includes a specific sequence, and the antibody includes (i) a VL domain including a CDR1 region, a CDR2 region, and a CDR3 region of a specific sequence, (ii) a VH domain including a CDR1 region, a CDR2 region, and a CDR3 region of a specific sequence, and (iii) an Fc region derived from human IgG1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to modified antibodies with improved effector function and / or pharmacokinetic properties, which are useful in the therapeutic treatment of various disorders, particularly inflammatory conditions. [Background technology]

[0002] Monoclonal antibodies have become increasingly important as therapeutic agents in clinical medicine over the past two decades. Over the years, efforts have focused on improving antibodies to reduce their potential immunogenicity, leading to humanized or fully human antibodies. Another approach aims to optimize antibodies by improving their effector functions. Direct effects are mediated by the variable antigen-binding region of the antibody, while indirect effects are mediated by the constant Fc region. Efforts to improve effector function have primarily focused on modulating the Fc region. Furthermore, improving the serum half-life of therapeutic antibodies is desirable; this could improve patient convenience by reducing the amount of antibody required and extending the treatment interval.

[0003] For therapeutic applications, immunoglobulin G (IgG) has become the preferred class of choice for several reasons: IgG is easily purified, relatively stable upon storage, can be administered intravenously, has a long biological half-life in vivo, and can participate in a range of biological effector functions, such as activation of complement-dependent cytotoxicity (CDC) and recruitment of effector cells through various Fc receptor interactions (antibody-dependent cellular cytotoxicity; ADCC). Among the five immunoglobulin classes, IgG exhibits the longest biological half-life due to its unique interaction with the IgG recycling receptor, the neonatal Fc receptor (FcRn). One of the receptor's known functions is to rescue IgG from catalytic degradation. Dissolved FcRn-Fc co-crystal structures have demonstrated that the interaction with Fc is via the IgG hinge-C. H 2-C HWe have shown that the FcRn-IgG interaction occurs in the -3 region of the IgG receptor. This interaction is strictly pH-dependent and occurs in endosomes at an acidic pH of 6.0–6.5. Bound IgG molecules are returned to the cell surface, where they are released into the circulation at a physiological pH of 7.4, while uncomplexed IgG molecules are destined for lysosomal degradation. This recycling is a mechanism for extending the half-life of IgG; therefore, modulation of the FcRn-IgG interaction allows for specific control of the serum half-life of gamma immunoglobulins and Fc fusion proteins.

[0004] Depending on the application, it may be desirable to extend or shorten the serum residence time of IgG. For therapeutic applications, a longer half-life is desirable because it allows for smaller doses and fewer injections. Several approaches to extend half-life have been investigated, including the use of polyethylene glycol (PEG), the creation of albumin or Fc fusion proteins, and the enhancement of FcRn-IgG interactions. PEGylated drugs have been available in clinics since the 1990s, and PEGylation is an established technique for extending drug residence time in the blood. Because human serum albumin (HSA) is also recycled by FcRn through a pH-dependent interaction, several albumin fusion proteins have been created to improve stability and half-life. Furthermore, antibody fragments fused to albumin or albumin-binding domains have demonstrated extended serum residence times in preclinical studies. The creation of Fc fusion proteins is another strategy for endowing proteins or peptides with properties similar to those of intact antibodies.

[0005] Fc region modifications that have been investigated are summarized in Saxena (2016) Frontiers in Immunology, Vol. 7, Article 580. Various Fc mutations are further described in WO 1998 / 023289 A1, WO 2000 / 042072 A2, WO 2010 / 106180 A2, and WO 2014 / 108198 A1.

[0006] There is an ongoing need for antibodies with improved effector functions and / or pharmacokinetics. Summary of the Invention

[0007] The inventors of the present application have found that certain mutations in the antibody Fc region increase the affinity of the antibody for FcRn at pH 6, while the affinity at pH 7.4 remains low. Antibodies with the mutations have improved pharmacokinetic properties. Furthermore, preferred antibody variants of the present invention have better T cell proliferation inhibitory activity than their respective parent wild-type antibodies.

[0008] Accordingly, the present invention relates to the subject matter defined in the following items [1] to

[91] : [1] A dissociation equilibrium constant (K) of less than 500 nM at pH 6 D ) and has a high affinity for human FcRn characterized by a K greater than 1 μM at pH 7.4. D An antibody comprising a TNFα-binding domain and an FcRn-binding site, and having low affinity for human FcRn, characterized by: wherein the amino acid sequence of the antibody comprises amino acids 380A and 434A. [2] The antibody according to item [1], wherein the antibody is obtained by substituting asparagine at position 434 with alanine (N434A). [3] The antibody according to item [1] or [2], wherein the antibody is obtained by substituting glutamic acid at position 380 with alanine (E380A). [4] The antibody according to any one of items [1] to [3], wherein the amino acid at position 307 of the amino acid sequence of the antibody is different from alanine. [5] The antibody according to any one of items [1] to [4], wherein the amino acid sequence of the antibody further comprises amino acid 307T. [6] The antibody according to any one of items [1] to [5], which comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 13. [7] The antibody according to any one of items [1] to [6], which has an affinity for human FcRn at pH 6 greater than the affinity of infliximab (IFX). [8] The high affinity for human FcRn at pH 6 has a dissociation constant K of less than 400 nM. D The antibody according to any one of items [1] to [7], characterized by: [9] The high affinity for human FcRn at pH 6 has a dissociation constant K of less than 300 nM. D The antibody according to any one of items [1] to [8], characterized by:

[10] The high affinity for human FcRn at pH 6 has a dissociation constant K of less than 200 nM. D The antibody according to any one of items [1] to [9], characterized by:

[11] The high affinity for human FcRn at pH 6 has a dissociation constant K of less than 150 nM. D The antibody according to any one of items [1] to

[10] , characterized by:

[12] The high affinity for human FcRn at pH 6 has a dissociation constant K in the range of 5 nM to 500 nM, or 10 nM to 400 nM, or 25 nM to 300 nM, or 50 nM to 200 nM, or 75 nM to 150 nM. D The antibody according to any one of items [1] to

[11] , characterized by:

[13] The K D The antibody according to any one of items [1] to

[12] , wherein the antibody is measured by surface plasmon resonance (SPR).

[14] The low affinity for human FcRn at pH 7.4 is greater than 10 μM D The antibody according to any one of items [1] to

[13] , characterized by:

[15] The K D The antibody according to any one of items [1] to

[14] , wherein the antibody is measured by SPR.

[16] Because of its very low affinity for human FcRn at pH 7.4, K D The antibody according to any one of items [1] to

[13] , wherein the value cannot be measured by SPR.

[17] K<200 pM for human TNFα D The antibody according to any one of items [1] to

[16] , which binds to

[18] K<100 pM for human TNFα D The antibody according to any one of items [1] to

[17] , which binds to

[19] K<50 pM for human TNFα D The antibody according to any one of items [1] to

[18] , which binds to

[20] K<25 pM for human TNFα D The antibody according to any one of items [1] to

[19] , which binds to

[21] K<10 pM for human TNFα D The antibody according to any one of items [1] to

[20] , which binds to

[22] The antibody according to any one of items [1] to

[21] , which is transported across a polarized cell monolayer from the apical side to the basolateral side.

[23] The antibody according to any one of items [1] to

[22] , which is transported across a polarized cell monolayer from the apical side to the basolateral side in greater amounts than a control antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2.

[24] The antibody according to any one of items [1] to

[23] , which is transported across a polarized cell monolayer from the apical side to the basolateral side in greater amounts than infliximab.

[25] The antibody according to item

[24] , wherein the amount of antibody transported across the polarized cell monolayer is greater than twice the amount of infliximab transported across the polarized cell monolayer.

[26] The antibody according to any one of items

[23] to

[25] , wherein the amount refers to the mass of antibody transported across the polarized cell monolayer within 4 hours.

[27] The antibody according to any one of items

[22] to

[26] , wherein the amount of antibody transported across the polarized cell monolayer is more than twice the amount of parent immunoglobulin transported across the polarized cell monolayer, and the parent immunoglobulin differs from the antibody only in that its Fc region contains only wild-type amino acids.

[28] The antibody according to any one of items

[22] to

[27] , wherein the amount of antibody transported across the polarized cell monolayer is more than twice the amount of parent immunoglobulin transported across the polarized cell monolayer, and the parent immunoglobulin differs from the antibody only in that the parent immunoglobulin contains amino acids 380E and 434N.

[29] The antibody according to any one of items [1] to

[28] , wherein in the presence of a 10-fold excess of a competing immunoglobulin, a greater percentage of the antibody than infliximab is transported across the polarized cell monolayer from the apical side to the basolateral side, and this percentage represents the total mass of immunoglobulin transported across the polarized cell monolayer.

[30] The antibody of item

[29] , wherein the percentage of the antibody that is transported across the polarized cell monolayer is more than twice the percentage of the parent immunoglobulin that is transported across the polarized cell monolayer, and the parent immunoglobulin differs from the antibody only in that the Fc region of the parent immunoglobulin has only wild-type amino acids.

[31] The antibody of item

[29] or

[30] , wherein the percentage of the antibody that is transported across the polarized cell monolayer is more than twice the percentage of the parent immunoglobulin that is transported across the polarized cell monolayer, and the parent immunoglobulin differs from the antibody only in that the parent immunoglobulin contains amino acids 380E and 434N.

[32] The antibody according to any one of items

[22] to

[31] , wherein the polarized cell monolayer is a monolayer of polarized T84 cells.

[33] K of CD64 less than 100 nM, preferably less than 10 nM D The antibody according to any one of items [1] to

[32] , which binds to

[34] CD32a(H) has a K of less than 10 μM D The antibody according to any one of items [1] to

[33] , which binds to

[35] CD32a(R) has a K of less than 10 μM D The antibody according to any one of items [1] to

[34] , which binds to

[36] CD32b has a K of less than 10 μM DThe antibody according to any one of items [1] to

[35] , which binds to

[37] K for CD16a(V) is less than 1000 nM, preferably less than 100 nM. D The antibody according to any one of items [1] to

[36] , which binds to

[38] K for CD16a(F) is less than 10 μM, preferably less than 1 μM D The antibody according to any one of items [1] to

[37] , which binds to

[39] K of less than 10 μM, preferably less than 1 μM, for CD16b(NA2). D The antibody according to any one of items [1] to

[38] , which binds to

[40] The antibody according to any one of items [1] to

[39] , which is capable of binding to human C1q.

[41] The antibody according to any one of items [1] to

[40] , which has complement-dependent cytotoxicity (CDC) of rabbit complement.

[42] The antibody according to any one of items [1] to

[41] , which has antibody-dependent cellular cytotoxicity (ADCC).

[43] The antibody according to any one of items [1] to

[42] , which is capable of inducing CD14+CD206+ macrophages.

[44] The antibody according to any one of items [1] to

[43] , which can induce CD14+CD206+ macrophages at a level equal to or greater than that of infliximab.

[45] The antibody according to any one of items [1] to

[44] , which is capable of suppressing T cell proliferation.

[46] The antibody according to any one of items [1] to

[45] , which can inhibit T cell proliferation to a degree equal to or greater than that of infliximab.

[47] The antibody according to any one of items [1] to

[46] , which is a non-fucosylated antibody or an antibody with reduced fucosylation.

[48] ​​(i) A V comprising a CDR1 region having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 5. Ldomain, and (ii) a V domain comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:6, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:7, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:8. H The antibody according to any one of items [1] to

[47] , comprising a domain.

[49] V having the amino acid sequence shown in SEQ ID NO: 9 H domain and V having the amino acid sequence set forth in SEQ ID NO: 10 L The antibody according to any one of items [1] to

[48] , comprising a domain.

[50] The antibody according to any one of items [1] to

[49] , comprising a light chain having the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 11.

[51] The antibody comprises (i) a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 16. L and (ii) a V domain comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 19. H The antibody according to any one of items [1] to

[47] , comprising a domain.

[52] V having the amino acid sequence shown in SEQ ID NO: 20 H domain and V having the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22 L The antibody according to item

[51] , comprising a domain.

[53] The antibody according to item

[51] or

[52] , comprising a light chain having the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 12.

[54] The antibody according to any one of items [1] to

[53] , wherein the antibody specifically binds to human TNFα.

[55] The antibody according to any one of items [1] to

[54] , wherein the antibody does not significantly bind to TNFβ.

[56] The antibody, (i) a dissociation constant (K) of less than 125 pM for human TNFα D ) to join; (ii) cross-reactive with rhesus monkey TNFα and cynomolgus monkey TNFα; (iii) has greater potency than infliximab as measured by the L929 assay; and / or (iv) a stoichiometric ratio of at least 2 (antibody:TNFα Trimer ) in human TNFα Trimer The antibody according to any one of items [1] to

[55] , which is capable of binding to

[57] K<1nM for TNFα from rhesus monkeys D The antibody according to any one of items [1] to

[56] , which binds to

[58] K<1nM for TNFα from cynomolgus monkeys D The antibody according to any one of items [1] to

[57] , which binds to

[59] The potency (relative potency) of an antibody for inhibiting TNFα-induced apoptosis is greater than 3 compared to the potency (relative potency) of infliximab determined in the L929 assay, and the relative potency is greater than the IC50 of infliximab in the L929 assay. 50 IC values ​​(ng / mL) of antibodies in the L929 assay 50 The antibody according to any one of items [1] to

[58] , wherein the ratio is a ratio of the antibody concentration to the antibody concentration (ng / mL).

[60] The antibody according to any one of items [1] to

[59] , wherein the melting temperature of the variable domain of the antibody in scFv format is at least 65°C as determined by differential scanning fluorimetry.

[61] The antibody according to any one of items [1] to

[60] , wherein the melting temperature of the variable domain of the antibody in scFv format is at least 68°C as determined by differential scanning fluorimetry.

[62] The antibody according to any one of items [1] to

[61] , which has a melting temperature of at least 70°C as measured by differential scanning fluorimetry.

[63] The antibody according to any one of items [1] to

[62] , which can block the interaction between human TNFα and TNF receptor I (TNFRI).

[64] The antibody according to any one of items [1] to

[63] , which can block the interaction between human TNFα and TNF receptor II (TNFRII).

[65] CD14 + The antibody according to any one of items [1] to

[64] , which can inhibit LPS-induced secretion of monocyte-derived interleukin-1β.

[66] IC to inhibit LPS-induced secretion of interleukin-1β 50 The antibody according to item

[65] , wherein the antibody has a IgG value of less than 1 nM.

[67] On a molar basis, IC for inhibiting LPS-induced secretion of interleukin-1β 50 The value is the IC 50 The antibody according to item

[66] , wherein the antibody has a value lower than the value.

[68] CD14 + The antibody according to any one of items [1] to

[67] , which can inhibit LPS-induced secretion of monocyte-derived TNFα.

[69] IC to inhibit LPS-induced secretion of TNFα 50 The antibody according to item

[68] , wherein the antibody has a IgG value of less than 1 nM.

[70] On a molar basis, IC for inhibiting LPS-induced secretion of TNFα 50 The value is the IC 50 The antibody according to item

[69] , wherein the antibody has a value lower than the value.

[71] The antibody according to any one of items [1] to

[70] , which is an immunoglobulin G (IgG), preferably an IgG1.

[72] A nucleic acid encoding the antibody according to any one of items [1] to

[71] .

[73] A vector or plasmid containing the nucleic acid of item

[72] .

[74] A cell containing the nucleic acid of item

[72] or the vector or plasmid of item

[73] .

[75] A method for producing the antibody according to any one of items [1] to

[71] , comprising culturing the cell according to item

[74] in a medium under conditions that allow expression of a nucleic acid encoding the antibody, and recovering the antibody from the cell or the medium.

[76] An antibody as defined in any one of items [1] to

[71] for use in a method for treating an inflammatory disease or a TNFα-related disorder.

[77] The antibody for use according to item

[76] , wherein the inflammatory disease is selected from the list of diseases and disorders listed in the "Disorders to be Treated" section below.

[78] The antibody for use according to item

[76] , wherein the inflammatory disease is an inflammatory disease of the gastrointestinal tract.

[79] The antibody for use according to item

[78] , wherein the inflammatory disease of the digestive tract is inflammatory bowel disease.

[80] The antibody for use according to item

[78] or

[79] , wherein the inflammatory disease of the gastrointestinal tract is Crohn's disease.

[81] The antibody for use according to item

[80] , wherein the Crohn's disease is selected from the group consisting of ileum, colon, ileocolonic, and / or isolated upper Crohn's disease (stomach, duodenum, and / or jejunum), including non-stricturing / non-penetrating, stricturing, permeating, and perianal disease behaviors, allowing for localization and any combination of any of the above disease behaviors.

[82] The antibody for use according to item

[78] or

[79] , wherein the inflammatory disease of the digestive tract is ulcerative colitis.

[83] The antibody for use according to item

[82] , wherein the ulcerative colitis is selected from the group consisting of ulcerative proctitis, sigmoiditis, proctosigmoiditis, left-sided colitis, pancolonic ulcerative colitis, and pouchitis.

[84] The antibody for use according to item

[78] or

[79] , wherein the inflammatory disease of the gastrointestinal tract is microscopic colitis.

[85] The antibody for use according to item

[76] , wherein the inflammatory disease is arthritis.

[86] The antibody for use according to item

[76] or

[85] , wherein the inflammatory disease is rheumatoid arthritis.

[87] The antibody for use according to any one of items

[76] to

[86] , wherein the method comprises orally administering the antibody to the subject.

[88] The antibody for use according to any one of items

[76] to

[87] , wherein the method comprises topically applying the antibody.

[89] A pharmaceutical composition comprising the antibody according to any one of items [1] to

[71] .

[90] A method for improving the transcytosis of an antibody against TNFα, comprising introducing the substitutions E380A and N434A into the amino acid sequence of the antibody.

[91] A method for increasing the plasma half-life of an antibody against TNFα, comprising introducing the substitutions E380A and N434A into the amino acid sequence of the antibody. [Brief explanation of the drawings]

[0009] [Figure 1] Potency of anti-TNFα antibody variants to neutralize human TNFα in the L929 assay. Dose-response curves for TNFα antibody-specific TP antibody and reference infliximab are shown. [Figure 2] Transport of anti-TNFα IgG variants across polarized T84 cells. Amounts of anti-TNFα antibody variants and IFX transported from the apical to the basolateral reservoir 4 hours after addition. Expressed as ng / cm². Error bars indicate SD of two to four individual monolayers. [Figure 3] Transport of anti-TNFα IgG variants across polarized T84 cells in the presence of excess myeloma IgG. Amounts of anti-TNFα antibody IFX and antibody variants transported from the apical to the basolateral reservoir in the presence of a 10-fold excess of human myeloma IgG 4 hours after addition. Expressed as ng / cm2. Error bars represent SD of 3-4 individual monolayers. [Figure 4] ADCC activity. ADCC induction by anti-TNFα antibody variants and wild-type antibody. [Figure 5] Binding to human C1q. Binding of IFX and anti-TNFα antibody variants to human C1q. Each concentration was assayed in duplicate. Error bars indicate SD. [Figure 6]Induction of CD14+CD206+ macrophages by each compound compared to IFX. Data are pooled from four independent experiments. Bars represent the mean, error bars represent SEM. [Figure 7] Inhibition of T cell proliferation by each compound compared to IFX. Data are pooled from three independent experiments. Bars represent the mean and error bars represent SEM. [Figure 8] Schematic representation of site-directed mutagenesis. [Figure 9] Schematic representation of the major N-glycan types attached to N297 of anti-TNFα antibody variants. The two predominant N-glycan profiles in the panel of anti-TNFα antibodies tested were 4GlcNac-1Fuc-3Man and 4GlcNac-1Fuc-3Man-1Gal, whereas the same biantennary structures, except lacking fucose, occurred for IgG variants produced in the presence of 2FF. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to an antibody capable of binding to TNFα and comprising an FcRn-binding site. According to the present application, an antibody comprises an FcRn-binding site if it can bind to FcRn, preferably human FcRn, at pH 6. Binding to FcRn at pH 6 can be measured by SPR, for example, as described in Example 4 of the present application. If the binding of an antibody to FcRn at pH 6 can be detected by SPR, then such an antibody has an FcRn-binding site. The antibody of the present invention has a dissociation equilibrium constant (K D ) at pH 6. The antibody has a high affinity for human FcRn, characterized by a K greater than 1 μM. D The antibody further has low affinity for human FcRn at pH 7.4, characterized by the following: The amino acid sequence of the antibody contains the amino acid alanine at position 380 and position 434 (EU numbering).

[0011] Throughout this specification, when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is typically used (Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is typically used (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (expressly incorporated herein by reference)). Unless otherwise specified, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise specified, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system (see, for example, WO 2006 / 073941).

[0012] antibody In the present application, the term "antibody" is used as a synonym for "immunoglobulin" (Ig), which is defined as a protein comprising all conventionally known antibodies and functional fragments thereof belonging to the IgG, IgM, IgE, IgA, or IgD class (or any subclass thereof). In the present invention, a "functional fragment" of an antibody / immunoglobulin is defined as an antigen-binding fragment or other derivative of a parent antibody that essentially maintains one or more properties of such parent antibody. An "antigen-binding fragment" or "antigen-binding domain" of an antibody / immunoglobulin is defined as a fragment that retains the antigen-binding region (e.g., the variable region of an IgG). The "antigen-binding region" of an antibody is typically found in one or more hypervariable regions of the antibody, i.e., the CDR-1, -2, and / or -3 regions. The antibody of the present invention may be part of a bifunctional or multifunctional construct.

[0013] Preferably, the antibody is a monoclonal antibody. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be produced using a variety of techniques known in the art, such as using hybridoma, recombinant, and phage display technologies, or a combination thereof (Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press 1988, Cold Spring Harbor, NY).

[0014] In other embodiments, including those directed to in vivo use of anti-TNFα antibodies in humans, chimeric, primatized, humanized, or human antibodies can be used. In preferred embodiments, the antibody is a human or humanized antibody, more preferably a monoclonal human or humanized antibody.

[0015] In another specific embodiment, the antibody of the present invention is an immunoglobulin, preferably an immunoglobulin G (IgG). The subclasses of the IgG of the present invention include, but are not limited to, IgG1, IgG2, IgG3, and IgG4. Preferably, the IgG of the present invention is subclass 1, 2, or 4, i.e., an IgG1, IgG2, or IgG4 molecule, respectively. Most preferably, the IgG of the present invention is subclass 1, i.e., an IgG1 molecule.

[0016] TNFα binding domain The TNFα-binding domain of the antibody of the present invention is not particularly limited and can be derived from any antibody capable of binding to TNFα.

[0017] Preferably, the antibody of the present invention specifically binds to TNFα. As used herein, an antibody "specifically recognizes" or "specifically binds" to human TNFα if it can distinguish between human TNFα and one or more reference molecules. Preferably, the IC for binding to each of the reference molecules is 0.01. 50 Values ​​are IC for binding to TNFα 50 The binding specificity is at least 1,000-fold greater than the value. In its most general form (and when no defining reference is given), "specific binding" refers to the ability of an antibody to distinguish between human TNFα and unrelated biomolecules, as measured, for example, by specificity assays known in the art. Such methods include, but are not limited to, Western blots and ELISA tests. For example, a standard ELISA assay can be performed. Typically, binding specificity is determined by using a set of about 3-5 unrelated biomolecules, such as milk powder, BSA, or transferrin, rather than a single reference biomolecule. In one embodiment, specific binding refers to the ability of an antibody to distinguish between human TNFα and human TNFβ.

[0018] The antibody of the present invention is V L Domains and V H Contains domain. V LA domain includes a CDR1 region (CDRL1), a CDR2 region (CDRL2), a CDR3 region (CDRL3), and framework regions. H A domain comprises a CDR1 region (CDRH1), a CDR2 region (CDRH2), a CDR3 region (CDRH3), and framework regions.

[0019] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) (Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat CDR definition applies only to CDR1, CDR2, and CDR3 (CDRL1, CDRL2, CDRL3, or L1, L2, L3) of the light chain variable domain and CDR2 and CDR3 (CDRH2, CDRH3, or H2, H3) of the heavy chain variable domain. However, as used herein, CDR1 (CDRH1 or H1) of the heavy chain variable domain is defined by the following residues (Kabat numbering): it begins at position 26 and ends before position 36.

[0020] In one embodiment of the invention, the antibody of the invention is an anti-TNFα antibody disclosed in any one of the currently filed international applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237, and PCT / EP2017 / 056227. In yet another embodiment of the invention, the antibody is an anti-TNFα antibody having a light chain variable domain and / or a heavy chain variable domain comprising complementarity determining regions (CDRs) having the amino acid sequences disclosed in the currently filed international applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237, and PCT / EP2017 / 056227.

[0021] In a preferred embodiment of the invention, the antibody is an anti-TNFα antibody having a light chain variable domain and / or a heavy chain variable domain comprising one or more CDRs having the amino acid sequences disclosed in PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 or PCT / EP2017 / 056227. In another preferred embodiment of the invention, the antibody is an anti-TNFα antibody having a light chain variable domain and a heavy chain variable domain comprising CDRs having the amino acid sequences disclosed in claim 2 of PCT / EP2017 / 056218, claim 2 of PCT / EP2017 / 056246, claim 2 of PCT / EP2017 / 056237 or claim 2 of PCT / EP2017 / 056227. In yet another preferred embodiment of the present invention, the anti-TNFα antibody is selected from the group consisting of anti-TNFα antibodies comprising the heavy chain variable domain amino acid sequence and / or the light chain variable domain amino acid sequence according to claim 4 of PCT / EP2017 / 056218, claims 5 and 6 of PCT / EP2017 / 056246, claims 5 and 6 of PCT / EP2017 / 056237, claim 4 of PCT / EP2017 / 056227, and combinations thereof. The disclosures of each of International Patent Applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237, and PCT / EP2017 / 056227 are incorporated herein in their entirety and form part of this application.

[0022] In certain embodiments, the antibodies of the invention comprise (i) a V CDR1 region having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:4, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:5. L domain, and (ii) a V domain comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:6, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:7, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:8. H Includes the domain.

[0023] In a more preferred embodiment, the antibody of the present invention comprises a V H In another more preferred embodiment, the antibody comprises a V domain having the amino acid sequence set forth in SEQ ID NO: 10. L Most preferably, the antibody of the present invention comprises (i) a V domain having the amino acid sequence set forth in SEQ ID NO: 9. H domain, and (ii) a V domain having the amino acid sequence set forth in SEQ ID NO: 10. L Includes the domain.

[0024] In another specific embodiment, the antibody of the invention comprises (i) a V CDR1 region having the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 16. L and (ii) a V domain comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 19. H Includes the domain.

[0025] In a more preferred embodiment, the antibody of the present invention comprises a V H In another more preferred embodiment, the antibody comprises a V domain having the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:22. L Most preferably, the antibody of the present invention comprises (i) a V domain having the amino acid sequence set forth in SEQ ID NO: 20. H domain, and (ii) a V having the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22. L Includes the domain.

[0026] The antibodies of the present invention have high affinity for human TNFα. D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present invention have a dissociation equilibrium constant of about 2 x 10 as measured using surface plasmon resonance (SPR) technology on a BIACORE instrument. -10Less than M, preferably 1.5x10 -10 Less than M, preferably 1.25x10 -10 Less than M, preferably 1x10 -10 Less than M, most preferably 7.5x10 -11 Less than M or 5x10 -11 The dissociation equilibrium constant (K D ) binds to human TNFα. D Measurements are performed as described in Example 1.

[0027] Modifications that affect affinity for FcRn The antibodies of the present invention comprise an amino acid sequence that differs from the native sequence of a wild-type antibody due to at least one "amino acid modification," as defined herein. The at least one amino acid modification affects the affinity of the antibody for human FcRn. Typically, the at least one amino acid modification increases the affinity of the antibody for human FcRn at pH 6. In one embodiment, the at least one amino acid modification increases the affinity of the antibody for human FcRn at pH 6 without substantially altering the affinity for human FcRn at pH 7.4. Preferably, the modified antibody has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, and preferably about 1 to about 5 amino acid substitutions, compared to the amino acid sequence of the wild-type or parent antibody. Preferably, the at least one amino acid modification is within the FcRn-binding site of the antibody. The antibody may have one or more amino acid modifications outside the FcRn-binding site of the antibody, which affect FcRn binding, for example, by structural changes. Amino acid modifications can be produced by methods known per se, for example by site-directed mutagenesis as described in "Antibody Engineering - Methods and Protocols", edited by Patrick Chames, 2nd ed., 2012, Chapter 31 (ISBN 978-1-61779-973-0).

[0028] The antibodies of the invention comprise the amino acid alanine at positions 380 and 434 (EU numbering). This is referred to herein as "380A" and "434A." The native amino acid at position 380 in unmodified human IgG antibodies is glutamic acid (E). The native amino acid at position 434 in unmodified human IgG antibodies is asparagine (N). Thus, the antibodies of the invention can be obtained by introducing the mutations E380A and N434A into the antibody. Preferably, the antibodies of the invention can be obtained or are obtained by substituting alanine for glutamic acid at position 380 and alanine for asparagine at position 434.

[0029] The remaining amino acid sequence of the Fc domain can be the same as the native amino acid sequence of a typical human IgG, but the amino acid sequence of the antibody can contain one or more mutations or substitutions relative to the native amino acid sequence of the Fc region of a native antibody, so long as the antibody retains TNFα-binding activity, FcRn-binding activity at pH 6.0, and one or more effector functions after the following modifications.

[0030] In a preferred embodiment, the Fc region of an antibody of the invention, including the hinge region, comprises or consists of the amino acid sequence set forth in SEQ ID NO:13.

[0031] In one embodiment, the heavy chain of the antibody of the invention has the amino acid sequence set forth in SEQ ID NO: 11. Preferably, the antibody further comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 1.

[0032] In another embodiment, the heavy chain of an antibody of the invention has the amino acid sequence set forth in SEQ ID NO: 12. Preferably, the antibody further comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 24.

[0033] In a preferred embodiment of the invention, the antibody of the invention is a non-fucosylated antibody or an antibody with reduced fucosylation.

[0034] As used herein, the term "antibody with reduced fucosylation" refers to an antibody in which less than 90% of the antibody's N-glycans are fucosylated.Methods for measuring the percentage of fucosylation are known in the art.Preferably, the percentage of fucosylation is measured as described in Example 10 of the present application.

[0035] In one embodiment, less than 75%, or less than 50%, or less than 25% of the N-glycans of the antibody are fucosylated. Most preferably, less than 10% of the N-glycans of the antibody are fucosylated. In a specific embodiment, the N-glycans of the antibody of the present invention do not contain any fucose.

[0036] Preferably, less than 90% of the N-glycans at N297 (EU numbering) of the antibody are fucosylated. In another embodiment, less than 75%, or less than 50%, or less than 25% of the N-glycans at N297 (EU numbering) of the antibody are fucosylated. Most preferably, less than 10% of the N-glycans at N297 (EU numbering) of the antibody are fucosylated.

[0037] In another embodiment, the N-glycan at position N297 of the antibody does not contain any fucose.

[0038] Nonfucosylated antibodies, sometimes referred to as afucosylated antibodies, can be produced by various methods. For example, synergistic knockdown of the genes for α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in CHO cells can be used to produce fully nonfucosylated monoclonal antibody variants with enhanced ADCC (see, e.g., Imai-Nishiya et al. (2007) BMC Biotechnol. 7, 84). Monoclonal antibodies completely lacking core fucose can be produced using a zinc finger nuclease (ZFN) method to cleave the FUT8 gene in the region encoding the catalytic core of α1,6-fucosyltransferase, thereby disrupting the corresponding enzyme function in CHO cells (see, e.g., Malphettes et al. (2010) Biotechnol. Bioeng. 106, 774-783).

[0039] Antibodies with reduced fucosylation can be generated by adding a decoy substrate, such as 2-deoxy-2-fluoro-2-fucose, to the culture medium to reduce the incorporation of fucose into IgG-Fc glycans (see, e.g., Dekker et al. (2016) Sci Rep 6:36964).

[0040] In another embodiment, the antibody of the present invention has a high sialic acid content. Increased sialylation can be achieved, for example, by co-transfection of cytidine monophosphate-sialic acid synthase (CMP-SAS), cytidine monophosphate-sialic acid transporter (CMP-SAT), and 2,3-sialyltransferase (see, e.g., Son et al. (2011) Glycobiology 21, 1019-1028).

[0041] Affinity for FcRn The affinity of the antibodies of the present invention for human FcRn at pH 6 is high. High affinity binding of the antibodies to human FcRn at pH 6 is demonstrated by a K D Preferably, the K value for high affinity binding at pH 6 is DFor example, the K value characterizing affinity at pH 6 is less than 400 nM, or less than 300 nM, or less than 200 nM. D Values ​​can range from 5 to 500 nM, or 10 to 400 nM, or 25 to 300 nM, or 50 to 200 nM, or 100 to 150 nM.

[0042] In a preferred embodiment, the affinity of the antibodies of the present invention for human FcRn at pH 6 is greater than the affinity of infliximab for human FcRn at pH 6.0.

[0043] The affinity of the antibody of the present invention for human FcRn is preferably measured by surface plasmon resonance (SPR) as described in Example 4 of the present application, for example.

[0044] The antibodies of the present invention typically have low affinity for human FcRn at pH 7.4. Low affinity is defined as a K D Preferably, the low affinity for human FcRn at pH 7.4 is characterized by a K value of greater than 2 μM, or greater than 5 μM, or greater than 10 μM. D It is characterized by a value.

[0045] In certain embodiments, the low affinity at pH 7.4 is D The value is too low to be measured by SPR.

[0046] In certain embodiments, (i) the K of binding of an antibody of the invention to human FcRn at pH 7.4 D (ii) K of binding to human FcRn at pH 6.0 D is at least 50. Preferably, the ratio is at least 100, or at least 150, or at least 200.

[0047] Functional properties of antibodies The antibodies of the invention are efficiently transported across polarized cell monolayers from the apical to the basolateral side. Typically, transport across polarized cell monolayers is greater than that of infliximab, where the amount of antibody transported is 100% of the mass of polarized cell monolayer / cm. 2 Relative to the amount of infliximab transported across the polarized cell monolayer, the amount of antibody transported across the polarized cell monolayer is at least 110%, preferably at least 120%, more preferably at least 130%, or at least 140%, or at least 150% (the amount of infliximab transported being 100%).

[0048] Furthermore, antibodies are specifically transported across polarized cell monolayers from the apical to the basolateral side in the presence of excess competing immunoglobulins, referred to herein as specific transport.

[0049] The percentage of the total mass of immunoglobulin transported across the polarized cell monolayer is greater than the percentage of infliximab transported across the polarized cell monolayer from apical to basolateral in the presence of a 10-fold excess of a competing immunoglobulin. The percentage of an antibody of the invention transported across the polarized cell monolayer in the presence of a 10-fold excess of an irrelevant immunoglobulin is at least 120%, or at least 130%, or at least 140%, or at least 150% relative to the percentage of infliximab transported across the polarized cell monolayer in the presence of a 10-fold excess of an irrelevant immunoglobulin (the amount of infliximab being 100%).

[0050] Preferably, the polarized cell monolayer is a monolayer of polarized T84 cells.The transcytotic transport assay mimicking process can be performed as described in Example 5 of the present application.

[0051] The antibodies of the invention bind to CD64, CD32a(H), CD32a(R), CD32b, CD16a(V), CD16a(F) and CD16b(NA2).

[0052] Antibodies of the invention typically have a K for CD64 of less than 100 nM, preferably less than 10 nM. D Combine with.

[0053] Antibodies of the invention typically have a K of less than 10 μM for CD32a(H). D Combine with.

[0054] Antibodies of the invention typically have a K of less than 10 μM for CD32a(R). D Combine with.

[0055] Antibodies of the invention typically have a K D Combine with.

[0056] Antibodies of the invention typically have a K for CD16a(V), e.g., less than 1 μM, preferably less than 500 nM, more preferably less than 100 nM. D Combine with.

[0057] Antibodies of the invention typically bind to CD16a(F), e.g., with a K of less than 10 μM, preferably less than 1 μM. D Combine with.

[0058] Antibodies of the invention typically bind to CD16b (NA2), e.g., with a K of less than 10 μM, preferably less than 1 μM. D Combine with.

[0059] The antibodies of the invention further bind to human C1q. Preferably, the strength of this binding of the antibodies of the invention to human C1q is at least as strong as the binding of infliximab to human C1q.

[0060] The antibodies of the present invention further have complement-dependent cytotoxicity (CDC) of rabbit complement.

[0061] The antibody of the present invention further comprises a CD14 + CD206 + It can induce macrophages, preferably at a level comparable to, the same as, or greater than that of infliximab.

[0062] The antibodies of the present invention can further suppress T cell proliferation, and the degree of suppression of T cell proliferation is preferably comparable to, the same as, or greater than the level of infliximab.

[0063] Pharmaceutical Compositions and Treatments Treatment of a disease includes treating a patient already diagnosed with any form of the disease at any clinical stage or symptom; delaying the onset, or progression, or exacerbation, or worsening of symptoms or signs of the disease; and / or preventing and / or reducing the severity of the disease.

[0064] A "subject" or "patient" to whom an anti-TNFα antibody is administered can be a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.), or a primate (e.g., monkey or human). In certain embodiments, the human is a pediatric patient. In other embodiments, the human is an adult patient.

[0065] Described herein are compositions comprising an anti-TNFα antibody and, optionally, one or more additional therapeutic agents, such as a second therapeutic agent described below. The composition is typically supplied as part of a sterile pharmaceutical composition that includes a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the desired method of administration to a patient).

[0066] Anti-TNFα antibodies can be administered to patients by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intrathecal, topically or locally, e.g., transmucosally. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Typically, anti-TNFα antibodies will be administered intravenously.

[0067] In a particularly preferred embodiment, the antibodies of the invention are administered orally. When administration is by the oral route, the antibody is preferably an IgG, most preferably an IgG1.

[0068] In typical embodiments, the anti-TNFα antibody is present in the pharmaceutical composition at a concentration sufficient to allow intravenous administration of 0.5 mg / kg to 20 mg / kg body weight. In some embodiments, concentrations of antibody suitable for use in the compositions and methods described herein include, but are not limited to, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, or a range between any of the foregoing values, e.g., 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, or 10 mg / kg to 18 mg / kg.

[0069] An effective dose of an anti-TNFα antibody can range from about 0.001 to about 750 mg / kg per single (e.g., bolus), multiple, or continuous administration, or can range to achieve a serum concentration of 0.01 to 5000 μg / mL per single (e.g., bolus), multiple, or continuous administration, or any effective range or value therein, depending on the condition being treated, the route of administration, and the age, weight, and condition of the subject. In the case of oral administration, serum concentrations can be very low or below the limit of detection. In certain embodiments, each dose can range from about 0.5 mg / kg body weight to about 50 mg / kg body weight or from about 3 mg / kg body weight to about 30 mg / kg body weight. The antibody can be formulated as an aqueous solution.

[0070] In a particularly preferred embodiment, the antibody of the present invention is administered orally. When administration is by the oral route, the antibody is preferably an IgG, most preferably an IgG1. When the antibody is administered orally, the daily dose of the antibody is typically in the range of about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.05 mg / kg body weight to about 50 mg / kg body weight, or about 0.1 mg / kg body weight to about 25 mg / kg body weight, or about 0.15 mg / kg body weight to about 10 mg / kg body weight, or about 0.16 mg / kg body weight to about 5 mg / kg body weight, or about 0.2 mg / kg body weight to about 2 mg / kg body weight, or about 0.2 mg / kg body weight to about 1 mg / kg body weight. Typically, a convenient dosage is 1 to 200 mg / day, preferably 5 to 100 mg / day or 10 to 50 mg / day.

[0071] Pharmaceutical compositions can be conveniently provided in unit dosage forms containing a predetermined amount of anti-TNFα antibody per dose. Such units can contain between 0.5 mg and 5 g, for example, but not limited to, 1 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 750 mg, 1000 mg, or any range between any two of the foregoing values, for example, 10 mg to 1000 mg, 20 mg to 50 mg, or 30 mg to 300 mg. Pharmaceutically acceptable carriers can take a wide variety of forms, depending, for example, on the condition being treated or the route of administration.

[0072] Determining an effective dose of anti-TNFα antibody, the total number of doses administered, and the length of treatment period is well within the capabilities of one of ordinary skill in the art and can be determined using standard dose escalation studies.

[0073] Therapeutic formulations of anti-TNFα antibodies suitable for the methods described herein can be prepared for storage as lyophilized formulations or aqueous solutions by mixing antibodies of the desired purity with any pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") commonly used in the art, such as buffers, stabilizers, preservatives, isotonicity agents, non-ionic surfactants, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to recipients at the dosages and concentrations used.

[0074] Buffers help maintain a pH in a range close to physiological conditions. They can be present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffers include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), citrate-phosphate buffers, succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumaric acid buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid- Examples of suitable buffers include disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconic acid buffers (e.g., gluconic acid-sodium gluconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium gluconate mixtures, etc.), oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactic acid buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

[0075] The pharmaceutical composition of the present invention may further comprise at least one salt, such as sodium chloride, preferably at a salt concentration in the range of 100 mM to 200 mM, for example, about 150 mM.

[0076] Preservatives can be added to retard microbial growth in amounts ranging from 0.2% to 1% (w / v). Suitable preservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and the like. Isotonicity agents, sometimes known as "stabilizers," can be added to ensure isotonicity of the liquid composition and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients that can serve a variety of functions, from bulking agents to additives that solubilize therapeutic agents or help prevent denaturation or adhesion to container walls. Typical stabilizers include polyhydric sugar alcohols (as listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols (cyclitols such as inositol) such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inisitol, galactitol, and glycerol; polyethylene glycol; amino acid polymers; urea, glutathione, and thiol. The stabilizer may be a sulfur-containing reducing agent such as lactic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, or sodium thiosulfate; a protein such as a low molecular weight polypeptide (e.g., a peptide of 10 residues or less), human serum albumin, bovine serum albumin, gelatin, or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; a monosaccharide such as xylose, mannose, fructose, or glucose; a disaccharide such as lactose, maltose, or sucrose; a trisaccharide such as raffinose; or a polysaccharide such as dextran. The stabilizer may be present in an amount ranging from 0.1 to 10,000 parts by weight per part by weight of active protein.

[0077] Non-ionic surfactants or detergents (also known as "wetting agents") can be added to aid in solubilizing the therapeutic agent, protect the therapeutic protein from agitation-induced aggregation, and allow the formulation to be exposed to stressed shear surfaces without denaturing the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), pluronic acid polyols, polyoxyethylene sorbitan monoethers (Tween®-20, Tween®-80, etc.). The non-ionic surfactant can be present in a range of about 0.05 mg / ml to about 1.0 mg / ml, or in a range of about 0.07 mg / ml to about 0.2 mg / ml.

[0078] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), protease inhibitors, and cosolvents.

[0079] The formulations herein may also include a second therapeutic agent in addition to the anti-TNFα antibody. Examples of suitable second therapeutic agents are provided below.

[0080] The administration schedule can vary from once a month to daily, depending on many clinical factors, such as the type of disease, the severity of the disease, and the patient's sensitivity to the anti-TNFα antibody. In certain embodiments, the anti-TNFα antibody is administered daily, twice a week, three times a week, every other day, every 5 days, once a week, every 10 days, every 2 weeks, every 3 weeks, every 4 weeks, or once a month, or within any range between any two of the above values, such as every 4 days to once a month, every 10 days to every 2 weeks, or 2-3 times a week.

[0081] The dosage of anti-TNFα antibody to be administered will vary depending on the particular antibody, the subject, as well as the nature and severity of the disease, the subject's health, the therapy (e.g., whether a second therapeutic agent is used), and the selected route of administration, and appropriate dosages can be readily determined by one of skill in the art.

[0082] Those skilled in the art will understand that the optimal amount and interval of each dosage of anti-TNFα antibody will be determined by the nature and severity of the condition being treated, the dosage form, administration route, and administration site, as well as the age and condition of the specific subject being treated, and the doctor will ultimately determine the appropriate dosage to be used. This dosage can be repeated as many times as necessary. If side effects occur, the amount and / or frequency of administration can be changed or reduced according to normal clinical practice.

[0083] Disorders to be treated The present invention relates to methods of treating or preventing a human TNFα-related disease in a subject, comprising administering to the subject an antibody as defined herein. The term "TNFα-related disorder" or "TNFα-related disease" refers to any disorder, onset, progression, or persistence of a symptom or disease state that requires the involvement of TNFα. Exemplary TNFα-related disorders include, but are not limited to, inflammatory chronic and / or autoimmune conditions generally, immune-mediated inflammatory diseases generally, inflammatory CNS diseases, inflammatory diseases affecting the eyes, joints, skin, mucous membranes, central nervous system, gastrointestinal tract, urinary tract, or lungs, uveitis conditions generally, retinitis, HLA-B27+ uveitis, Behcet's disease, dry eye syndrome, glaucoma, Sjogren's syndrome, diabetes mellitus (including diabetic neuropathy), insulin resistance, arthritic conditions generally, rheumatoid arthritis, inflammatory bowel disease, and inflammatory conditions of the urinary tract. Osteoarthritis, reactive arthritis and Reiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, amyotrophic lateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidney disease, cystitis, psoriasis (including psoriatic arthritis), hidradenitis suppurativa, subcutaneous panniculitis, pyoderma gangrenosum, SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis and osteitis), acne, Sweet's syndrome, pemphigus, Crohn's disease (including extraintestinal manifestations), ulcerative colitis, bronchial asthma, hypersensitivity pneumonitis, total urinary tract infections, ... Synthetic allergies, allergic rhinitis, allergic sinusitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, Wegener's granulomatosis, Kawasaki syndrome, giant cell arteritis, Churg-Strauss vasculitis, polyarteritis nodosa, burns, graft-versus-host disease, host-versus-graft reaction, rejection episodes after organ or bone marrow transplantation, general systemic and local conditions of vasculitis, systemic and cutaneous lupus erythematosus, polymyositis and dermatomyositis, scleroderma, preeclampsia, acute and chronic pancreatitis, viral hepatitis, alcoholic hepatitis, post-operative inflammation such as after eye surgery (e.g., cataract (eye lens replacement) or glaucoma surgery), joint surgery (including arthroscopic surgery), surgery on joint-related structures (e.g., ligaments), oral and / or dental surgery, minimally invasive cardiovascular procedures (e.g., PTCA, atherectomy, stent placement), laparoscopic and / or endoscopic intraperitoneal and gynecological procedures, endoscopic urological procedures (e.g., prostate surgery, ureteroscopy, cystoscopy, interstitial cystitis), or peri- and post-operative inflammation (prophylaxis) in general,These include bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, pustular dermatitis, cerebral malaria, hemolytic uremic syndrome, allograft rejection, otitis media, snakebite, erythema nodosum, myelodysplastic syndrome, primary sclerosing cholangitis, seronegative spondyloarthropathy, autoimmune hemolytic anemia, orofacial granulomatosis, vegetative suppurative stomatitis, aphthous stomatitis, geographic tongue, migratory stomatitis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Bell's palsy, Creutzfeldt-Jakob disease, as well as neurodegenerative conditions in general.

[0084] Cancer-related osteolysis, cancer-related inflammation, cancer-related pain, cancer-related cachexia, bone metastases, acute and chronic types of pain whether due to central or peripheral actions of TNFα and whether they are classified as inflammatory, nociceptive or neuropathic types of pain, sciatica, low back pain, carpal tunnel syndrome, complex regional pain syndrome (CRPS), gout, post-herpetic neuralgia, fibromyalgia, regional pain states, chronic pain syndromes due to metastatic tumors, dysmenorrhea.

[0085] Specific disorders to be treated include arthritic conditions in general, rheumatoid arthritis, osteoarthritis, reactive arthritis, juvenile arthritis, psoriasis such as psoriatic arthritis, inflammatory bowel diseases such as Crohn's disease, ulcerative colitis such as proctitis, sigmoiditis, proctosigmoiditis, left-sided colitis, extensive colitis and pancolitis, indeterminate colitis, microscopic colitis such as collagenous and lymphocytic colitis, colitis in connective tissue diseases, diversion colitis, colitis in diverticular disease, eosinophilic colitis, and pouchitis.

[0086] Most preferably, the antibodies of the present invention are used to treat inflammatory bowel diseases, particularly Crohn's disease, ulcerative colitis, or microscopic colitis. Crohn's disease can be ileal, colonic, ileocolonic, or isolated upper Crohn's disease (stomach, duodenum, and / or jejunum), including non-stricturing / non-penetrating, stricturing, penetrating, and perianal disease behaviors, and any combination of any of the above localizations and disease behaviors is possible. Ulcerative colitis can be ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolonic ulcerative colitis, and pouchitis.

[0087] Combination Therapy and Other Aspects Preferably, patients treated with anti-TNFα antibodies are also treated with other conventional drugs. For example, patients with inflammatory bowel disease, especially those with moderate to severe disease, are usually treated with mesalazine or its derivatives or prodrugs, corticosteroids (oral or intravenous) such as budesonide or prednisolone, immunosuppressants such as azathioprine / 6-mercaptopurine (6-MP) or methotrexate, cyclosporine, or tacrolimus. Other drugs that can be co-administered to patients include other anti-TNFα antibodies (e.g., infliximab, adalimumab, etanercept, certolizumab pegol, golimumab), integrin antagonists (e.g., natalizumab, vedolizumab), anti-IL-23 antibodies (e.g., MEDI2070), anti-β7 antibodies (e.g., etrolizumab), JAK inhibitors of the JAK / STAT pathway (e.g., tofacitinib), etc. Additional medications that may be co-administered to the patient include immunosuppressants (e.g., azathioprine / 6-MP or methotrexate or oral cyclosporine) to maintain stable, longer-term remission. Yet another aspect of the present invention is the use of an anti-TNFα antibody as defined herein above to reduce inflammation.

[0088] Yet another aspect of the present invention is an anti-TNFα antibody as defined herein above for use in reducing inflammation in a patient suffering from an inflammatory condition.

[0089] A further aspect of the invention is a method for treating an inflammatory condition, which comprises administering to a patient in need thereof an effective amount of an anti-TNFα antibody as defined herein above, the inflammatory condition preferably being one of the above conditions.

[0090] A further aspect of the present invention is a method for preventing an inflammatory condition, which comprises administering to a patient in need thereof an effective amount of an anti-TNFα antibody as defined herein above, the inflammatory condition preferably being one of the above conditions.

[0091] Yet another aspect of the present invention is a method for improving the transcytosis of an antibody against TNFα, comprising introducing the substitutions E380A and N434A into the amino acid sequence of the antibody to obtain a modified antibody with improved transcytosis, the modified antibody preferably being an antibody as described herein above.

[0092] Yet another aspect of the present invention is a method for extending the plasma half-life of an antibody against TNFα, comprising introducing substitutions E380A and N434A into the amino acid sequence of the antibody to obtain a modified antibody with an extended plasma half-life. The modified antibody is preferably an antibody as described hereinabove. The plasma half-life may be extended by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to the plasma half-life of the unmodified antibody (i.e., the parent antibody lacking the substitutions E380A and N434A). [Table 1]

[0093] Example Antibody variants Several variants of the anti-TNFα antibody were generated by introducing substitutions into the Fc region of the antibody amino acid sequence. Mutations were introduced using established site-directed mutagenesis methods. Briefly, mutations were introduced by PCR. A forward primer was designed to contain the desired mutation, and a reverse primer was simultaneously designed so that the 5' ends of the two primers annealed back-to-back (but without overlapping) (Figure 8). PCR was performed for 25 cycles (98°C for 10 seconds, 64°C for 30 seconds, and 72°C for 3 minutes). Before subjecting the PCR products to agarose gel analysis, the unmutated PCR template was removed from the PCR product pool using the restriction enzyme DpnI. After gel purification of the PCR product, blunt ends were ligated to obtain a circularized plasmid, which was then transformed into competent E. coli cells. After overnight incubation, several colonies were picked, and the plasmid DNA was isolated and sequenced to confirm the incorporation of the mutations.

[0094] A nonfucosylated variant was generated by adding 0.15 mM of the decoy substrate 2-deoxy-2-fluoro-2-fucose (Dekkers et al. (2016) Sci Rep 6:36964), which significantly reduced the incorporation of fucose into IgG-Fc glycans, as shown in Example 10 below. [Table 2]

[0095] Example 1. Affinity for TNFα method: Affinity for TNFα was measured by Biacore. CM5 chips were prepared using the standard amine-immobilized Biacore procedure. Upon insertion of the CM5 chip, the system was primed and then normalized with BIA normalization solution (Biacore Preventative Maintenance Kit 2). The chip was added to the system with PBS-T running buffer; prior to immobilization, the chip surface was primed with three injections of 50 mM NaOH. Protein A was immobilized on the chip surface. For this, the protein was diluted to 5 μg / mL in 10 mM acetate buffer at pH 4.5 and injected, generating a binding response of approximately 1,000 RU across all four flow cells. Three 15-second washes with 50 mM NaOH were performed to remove noncovalently bound material from all chip flow cells. On the Protein A chip, antibody was captured in flow cells 2 and 4, while flow cells 1 and 3 were used for baseline subtraction. The test antibody was diluted to 10 nM in PBS-T, and 2.5–7.5 μL was injected to obtain 120 RU of capture antibody. Following the supplier's instructions, the analyte TNFα was prepared at 500 μg / mL in water and further diluted in the running buffer, phosphate-buffered saline Tween-20 (PBS-T). Single-cycle kinetics was used to estimate steady-state affinity. For each single-cycle analysis cycle, a titration of five analyte concentrations was injected over the ligand, followed by measurement of complex dissociation. The surface was regenerated with glycine pH 1.7. A double reference method was employed, where data from the ligand-bound capture surfaces (fc2 and 4) were subtracted from the reference surfaces (fc1 and 3, respectively) with no ligand captured. Buffer blank injections were performed every 3–4 cycles and then subtracted from the analyte injection cycles to correct for small changes in the ligand capture surface. Replicate injections of analyte at the beginning and end of each analytical run were used to investigate sample degradation or changes in instrument performance. All analyses were performed at 25°C, and sample racks were incubated at 10°C throughout the experiment. Each experiment was performed at least three times. A one-to-one binding model was used to fit the resulting kinetic data.

[0096] result: All antibodies showed similar binding kinetics to TNFα, and none of the introduced modifications resulted in major changes in the antigen-binding region. [Table 3]

[0097] Example 2. Efficacy method: L929 cells were incubated with 0.25 ng / mL TNFα and 1 μg / well actinomycin D in the presence of serial dilutions of anti-TNFα antibody variants. After 20 hours of incubation at 37°C / 5% CO2, the proliferative response was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling reagent (phenazine ethosulfate, PES). MTS was converted to a formazan product by dehydrogenases present in metabolically active cells. The amount of formazan product, measured by absorbance at 492 nm, was directly proportional to the number of viable cells in culture.

[0098] result: The results are shown in Figure 1. Introduction of mutations into the Fc region of the anti-TNFα antibody did not affect potency.

[0099] Example 3. Affinity for Fcγ receptors (CD64, CD32a, CD32b, CD16a, CD16b) method: Affinity for FcγR was measured by Biacore. CM5 chips were prepared using the standard amine-immobilized Biacore procedure. Upon insertion of the CM5 chip, the system was primed and then normalized with BIA normalization solution (Biacore Preventative Maintenance Kit 2). The chip was added to the system with PBS-T running buffer; prior to immobilization, the chip surface was primed with three injections of 50 mM NaOH. FcγR was immobilized on the chip surface using a His-tag capture system. Anti-His-tag chips were prepared according to the Biacore kit instructions, and approximately 12,000 RU of antibody was deposited on all four flow cells. Three 30-second washes with 10 mM glycine, pH 1.5, were performed to remove noncovalently bound material from all chip flow cells. Fcγ receptors were diluted in PBS-T to a range of 0.5–2 μg / mL, and 2.5–5.0 μL was injected onto the chip, producing capture levels of 60–200 RU. Antibodies were diluted in PBS-T prior to analysis. Single-cycle kinetics were used to estimate steady-state affinity. For each single-cycle analysis cycle, a titration of five antibody concentrations was injected over the FcγR ligand, followed by measurement of complex dissociation. The surface was regenerated for the anti-His capture surface using the recommended solution, 10 mM glycine pH 1.5. A double reference method was employed, where data from the ligand-bound capture surfaces (fc2 and 4) were subtracted from the reference surfaces with no ligand captured (fc1 and 3, respectively). A buffer blank injection was performed after each antibody titration cycle and then subtracted from the analyte injection cycle to correct for small changes in the ligand capture surface. All analyses were performed at 25°C, and the sample rack was incubated at 10°C throughout the experiment. Each experiment was performed at least three times.

[0100] result: Binding to CD64 was unaffected by the genetically engineered anti-TNFα antibody. The introduction of mutations did not affect affinity for CD32a(H), CD32a(R), or CD32b. However, antibody Ab-AA-2FF showed a 4.7-fold increase in affinity for CD16a(V). Notably, the nonfucosylated antibody Ab-AA-2FF also improved binding to the low-affinity CD16a receptor and CD16b. [Table 4] [Table 5]

[0101] Example 4. Affinity for FcRn method: SPR was performed on a Biacore 3000 instrument using a CM5 sensor chip coupled with an anti-TNFα IgG1 antibody (approximately 500 resonance units (RU)) using amine coupling chemistry as described by the manufacturer. Coupling was performed by injecting 2.0 μg / mL of each protein in 10 mM sodium acetate, pH 4.5, using an amine coupling kit (GE Healthcare). HBS-P buffer pH 7.4 (10 mM HEPES, 150 mM NaCl, 0.005% surfactant P20) or phosphate buffer pH 6.0 (67 nM phosphate buffer, 150 mM NaCl, 0.005% Tween 20) was used as the running and dilution buffers. Binding kinetics were determined by injecting titration amounts (1000–31.2 nM) of monomeric His-tagged human FcRn (hFCRn) over the immobilized antibody at pH 7.4 or pH 6.0. All SPR experiments were performed at 25°C with a flow rate of 40 ul / min. Binding data were zero-adjusted and reference cell values ​​were subtracted. Binding kinetics were determined using the Langmuir 1:1 ligand binding model provided by BIAevaluation software (version 4.1).

[0102] result: The results showed that the wild-type antibody Ab-wt bound to hFcRn in a strictly pH-dependent manner. The engineered antibody variants had higher affinity for FcRn at pH 6.0 but retained their pH dependence and did not bind to the receptor at pH 7.4. All antibody variants showed improved binding to FcRn compared to infliximab, which contains the wild-type IgG1 Fc region. [Table 6]

[0103] Example 5. Transcytosis method: Transwell filters (1.12 cm) with 0.4 μm-sized collagen-coated polytetrafluoroethylene (PTFE) membranes 2 ) were incubated overnight in complete growth medium, followed by 1.0x10 cells per well. 6 T84 cells were seeded onto the cells. Transepithelial electrical resistance (TEER) was monitored daily using a Millicell-ERS-2 volt-ohm meter. After the cultures were grown for 4-5 days, the resistance reached approximately 1000-1300 Ω x cm. 2The monolayers reached confluence with a TEER value of 0.01. Prior to the experiment, the monolayers were starved in Hank's balanced salt solution (HBSS) for 1 hour. Then, 400 nM of antibody variants or IFX alone, or together with 4000 nM of human myeloma IgG with irrelevant specificity, were added to the apical transwell chamber. Samples were collected from the basolateral reservoir at 0 and 4 hours after addition. The antibody concentration in the basolateral reservoir was measured by ELISA. Briefly, 96-well Maxisorp plates were coated overnight with recombinant TNFα or goat-derived anti-human Fc-specific antibodies (both diluted to 1 μg / ml in PBS). The plates were then blocked with 4% nonfat milk in PBS for 2 hours at room temperature, followed by four washes with 0.05% Tween 20 in PBS. Samples collected during the transcytosis experiment were added to the wells, incubated for 2 hours at room temperature, and then washed as described above. The captured antibody variants, IFX, or total IgG were detected using alkaline phosphatase (ALP)-conjugated goat anti-human Fc-specific antibodies. Binding was visualized by adding 100 μl of ALP-substrate, and the absorbance spectra were recorded at 405 nm. The amounts of transferred antibody variants, IFX, and total IgG were calculated from the calibration curves of each individual antibody variant.

[0104] Transcytosis of antibody variants across polarized human epithelial cells result: The transcytosis of genetically engineered anti-TNFα antibody variants across cell monolayers was tested and compared with IFX, another human IgG1 anti-TNFα antibody. The results are shown in Figure 2. Compared to IFX, the IgG1 antibody with a wt Fc region, Ab-AA, was transported approximately 1.8-fold more efficiently. A similar significant increase in transport of Ab-AA was also observed for the nonfucosylated version, Ab-AA-2FF.

[0105] Transcytosis of antibody variants across polarized human epithelial cells in the presence of competing IgG result: When anti-TNFα antibody variants were incubated with a 10-fold excess of human myeloma IgG 4 hours after addition, the total amount of immunoglobulin transported across polarized T84 cell monolayers from the apical to the basolateral reservoir was comparable for all antibodies. However, the increased affinity for FcRn at pH 6.0 resulted in a significantly higher percentage of specific anti-TNFα transport across the cell monolayer, even in the presence of excess competing human IgG with irrelevant specificity. This result is shown in Figure 3.

[0106] Example 6. ADCC method: The ADCC Reporter Bioassay Core Kit from Promega was used. Briefly, 1x10 mTNFα CHO-K1 target cells were cultured at 1x10 5 100 μL per well was seeded into white (clear bottom) tissue culture dishes at 100 μL / mL. Plates were incubated overnight at 37°C / 5% CO2. On day 2, 95 μL of assay medium was removed and 25 μL of 3x10 cells were added. 6 1 / mL of genetically modified Jurkat effector cells was substituted. The plate was then incubated at 37°C / 5% CO2 for 6 hours. Toward the end of the incubation, BioGlo™ Reagent was prepared. The plate was equilibrated at room temperature for 10-20 minutes, after which 75 μL of BioGlo™ Reagent was added per well. After 5-10 minutes of incubation, luminescence was measured in the dark. The data were fitted using a 4-PL model.

[0107] result: The results (see Figure 4) showed that all anti-TNFα antibodies induced ADCC, although with different potencies. Compared to the wild-type antibody Ab-wt, Ab-AA showed similar ADCC activity, whereas the nonfucosylated antibody variant Ab-AA-2FF had significantly improved ADCC.

[0108] Example 7. C1q Binding method: ELISA was performed using 96-well Maxisorp plates coated with human TNFα diluted to 1 μg / mL in PBS. After overnight incubation at 4°C, the plates were blocked for 1 hour with 4% nonfat milk in PBS and washed four times with 0.05% Tween 20 in PBS (PBS-T). A titrated amount of anti-TNFα IgG antibody was then diluted in PBS-T, added, and incubated for 1 hour at room temperature. After washing with PBS-T, human C1q (0.5 μg / mL) was diluted in 0.1 M veronal buffer (0.25 mM CaCl2 and 0.8 mM MgCl2, pH 7.2) and added to the wells for 1 hour. Subsequently, the wells were washed as described above, and rabbit anti-human C1q diluted 1:5000 in PBS-T was added to the wells and incubated for 1 hour. After washing, donkey HRP-conjugated anti-rabbit IgG diluted 1:5000 in PBS-T was added. Subsequently, the wells were washed, and 100 μL of 3,3',5,5'-tetramethylbenzidine substrate was added to each well. Absorbance was measured at 620 nm using a Sunrise spectrophotometer.

[0109] result: The anti-TNFα antibody variants were captured by human TNFα before human C1q was added. The results (see Figure 5) showed that the antibodies bound to C1q, but with different binding strengths. The ranking of binding, from strongest to weakest, was as follows: Ab-AA=IFX>Ab-AA-2FF.

[0110] Example 8. Induction of regulatory macrophages method: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy buffy coats by Ficoll density gradient centrifugation. Equal numbers of cells from two individual donors were mixed, and 2 x 10 cells of the mixture were collected. 5Cells were seeded into 96-well plates in a total volume of 100 μL per well. Cells were incubated at 37°C / 5% CO2 for 48 hours. After 48 hours, anti-TNFα antibody variants or IFX were added to reach a final concentration of 10 μg / mL. Each compound was added in five or six replicates. The final volume was 150 μL per well. Human serum IgG1 (Sigma #I5154) was used as a control. After compound addition, the mixed lymphocyte reaction (MLR) was cultured for an additional 4 days at 37°C / 5% CO2. Afterwards, the plate was washed with PBS / 5mM EDTA (PBS / EDTA) and incubated with 50 μL per well of PBS / EDTA for 20 minutes at room temperature. The plate was centrifuged and the liquid was removed. Antibodies were diluted in PBS / EDTA (anti-CD14-PE, anti-CD206-APC, both diluted 1:10). Cells were resuspended in 50 μL of antibody solution and incubated for 20 minutes at room temperature. Afterwards, cells were washed with PBS / EDTA and resuspended in 50 μL of PBS / EDTA. Stained samples were analyzed on a FACS Fortessa using FACSDiva software. Analysis was performed using FlowJo software.

[0111] result: The induction of regulatory macrophages was analyzed in four independent MLRs and was successful in all experiments (comparing IFX to the IgG control). The results are shown in Figure 6. The level of induction by IFX may vary between experiments due to the fact that each experiment was performed using a different donor with inter-individual variability. All tested anti-TNFα antibody variants inhibited CD14 + CD206 + Regulatory macrophages were induced with little variation between compounds. Ab-AA-2FF induced significantly more regulatory macrophages than IFX.

[0112] Example 9. Inhibition of T cell proliferation method: PBMCs were isolated from healthy buffy coats by Ficoll density gradient centrifugation. Cells from two individual donors were mixed in equal numbers, and 2 x 10 cells of the mixture were collected. 5Cells were seeded in a 96-well plate in a total volume of 100 μL / well. The cells were incubated at 37°C / 5% CO2 for 48 hours. After 48 hours, anti-TNFα antibody variants or IFX were added to reach a final concentration of 10 μg / mL. Each compound was added in 5 or 6 replicates. The final volume was 150 μL / well. Human serum IgG1 (Sigma #I5154) was used as a control. After compound addition, the mixed lymphocyte reaction (MLR) was cultured for an additional 2 days at 37°C / 5% CO2. Afterwards, tritiated thymidine ( 3 H thymidine (0.5 microcuries / well) was added to the cultures. Cultures were further incubated at 37°C / 5% CO2 for 18 hours. Samples were collected using a Microbeta Filtermat96 cell harvester and analyzed using a Microbeta Microplate Counter equipped with a single detector. Samples were counted for 10 seconds / well and converted to counts per minute (cpm).

[0113] result: Inhibition of T cell proliferation was measured in three independent MLRs, and success was defined as induction of suppression by IFX as a positive control. The level of suppression by IFX in individual experiments may vary, likely due to variability in regulatory macrophage induction. In each experiment, the potential of anti-TNFα antibody variants to suppress T cell proliferation was calculated relative to the positive control IFX. The antibody Ab-AA-2FF demonstrated significantly enhanced suppression compared to IFX, while Ab-AA showed significantly less suppression of T cell proliferation than IFX (see Figure 7).

[0114] Example 10. Analysis of N-glycans method: Fifty microliters of each IgG variant (1 mg / ml) was centrifuged at 13,000 x g for 10 minutes, followed by the addition of 1 μg of trypsin dissolved in 100 μl of 50 mM ammonium bicarbonate (pH 7.8) and incubation overnight at 37°C. The mixture was then centrifuged at 13,000 x g for 10 minutes in a centrifuge. The flow-through was transferred to an Eppendorf tube and dried in a SpeedVac (Heto Maxi dry). The dried sample was dissolved in 20 μl of 1% formic acid, sonicated for 30 seconds, and centrifuged at 16,100 x g for 10 minutes. Each sample was then transferred to a new vial, and reversed-phase (C18) nano-online liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of proteolytic peptides was performed using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, USA). 5 μL of peptide solution was injected onto the extraction column, and peptides were eluted from the extraction column to the analytical column in backflush mode. The mobile phase consisted of acetonitrile and mass spectrometry-grade water (both containing 0.1% formic acid). Chromatographic separation was performed using a binary gradient from 3 to 50% acetonitrile in water over 60 min at a flow rate of 0.3 μL / min. The LC system was coupled to a Q exactive hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific, USA) via a nanoelectrospray ion source. Peptide samples were analyzed by higher-energy collision dissociation (HCD) fragmentation using a normalized collision energy of 20. One orbitrap survey scan was acquired in the mass range of m / z 300–2000, followed by MS / MS acquisition of the 10 most intense ions in the orbitrap.

[0115] Data analysis was performed using Xcalibur v2.0. MS / MS spectra of all N-glycopeptides were extracted using an oxonium ion search: 204.086 (N-acetylhexosamine) and 366.1388 (N-acetylhexosamine-hexose). The glycan structure and peptide mass of IgG were detected using HCD fragmentation with a normalized collision energy of 20. The extracted ion chromatogram of the target glycolipid (EEQYNSTYR of IgG1) was extracted with an accuracy of 10 ppm, and the corresponding MS / MS spectrum was manually verified. Peptide sequences were detected using HCD fragmentation with a normalized collision energy of 35, and it was verified that the peptide mass corresponded to the correct peptide sequence. The area under the curve of all extracted glycolipids was calculated, and the percentage ratio of each glycoform was determined.

[0116] result: In the case of Ab-AA, two N-glycan types, i.e., 4GlcNac-1Fuc-3Man and 4GlcNac-1Fuc-3Man-1Gal, predominated, representing over 90% of the total N-glycan pool. Both predominant N-glycan types contained core fucose. To generate a "nonfucosylated" version of Ab-AA (Ab-AA-2FF), we used the decoy substrate 2-deoxy-2-fluoro-1-fucose (2FF). MS mapping of this antibody revealed that this strategy successfully reduced fucose incorporation to a significant extent, with a detectable drop in fucose content from over 90% to less than 10%. The predominant N-glycan types after treatment were identical to those generated in the absence of 2FF, except that these structures lacked fucose (see also Figure 9). [Table 7]

Claims

1. Dissociation equilibrium constant (K) of less than 500 nM at pH 6 Ｄ ) and further have a high affinity for human FcRn characterized by a K of greater than 10 μM at pH 7.

4. Ｄ An antibody comprising a TNFα-binding domain and an FcRn-binding site, which has no or low affinity for human FcRn, characterized by: the amino acid sequence of the antibody is containing the amino acids 380A (EU numbering) and 434A (EU numbering), The antibody (i) a V comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 16; Ｌ domain, (ii) a V comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 19; Ｈ domain, and (iii) an Fc region derived from human IgG1 an antibody,

2. The antibody of claim 1 , wherein the amino acid sequence of the antibody further comprises amino acid 307T (EU numbering).

3. The antibody of claim 1 or 2, having an affinity for human FcRn at pH 6 that is greater than the affinity of infliximab.

4. The high affinity for human FcRn has a dissociation constant K of less than 300 nM. Ｄ The antibody according to any one of claims 1 to 3, characterized in that:

5. K<100 pM for human TNFα Ｄ The antibody according to any one of claims 1 to 4, which binds to

6. The antibody of any one of claims 1 to 5, which is transported across polarized cell monolayers from the apical to the basolateral side in greater amounts than infliximab.

7. 7. The antibody of claim 6, wherein the amount of antibody transported across the polarized cell monolayer is more than twice the amount of parent immunoglobulin transported across the polarized cell monolayer, and wherein the parent immunoglobulin differs from the antibody only in that the parent immunoglobulin contains amino acids 380E (EU numbering) and 434N (EU numbering).

8. 8. The antibody of any one of claims 1 to 7, wherein in the presence of a 10-fold excess of a competing immunoglobulin, a greater percentage of the antibody than infliximab is transported from the apical to the basolateral side across a polarized cell monolayer, said percentage representing the total mass of immunoglobulin transported across the polarized cell monolayer.

9. The antibody of any one of claims 1 to 8, which is a non-fucosylated antibody.

10. A nucleic acid encoding the antibody according to any one of claims 1 to 9.

11. An antibody according to any one of claims 1 to 9 for use in the treatment of an inflammatory condition.

12. The antibody for use according to claim 11, wherein the inflammatory condition is an inflammatory disease of the gastrointestinal tract.

13. The antibody for use according to claim 11 , wherein the treatment comprises orally administering an effective amount of the antibody.

14. The antibody for use according to claim 11 or 12, wherein the antibody is applied topically.

15. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 9.

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