Antibody variants

Abstract

The present invention relates to antibodies that bind to TNF [alpha] and exhibit modified FcRn binding. The antibodies of the invention have good effector function and / or pharmacokinetic properties.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201880060497.2, application date September 11, 2018, and invention name “Antibody Variants”. Technical Field

[0002] The present invention relates to modified antibodies with altered effector functions and / or altered pharmacokinetic properties. The antibodies can be used to treat various diseases, particularly inflammatory diseases. Background Art

[0003] Over the past 20 years, monoclonal antibodies have become increasingly important as therapeutic agents in clinical medicine. Over the years, efforts to improve antibodies have focused on reducing their potential immunogenicity, resulting in humanized or fully humanized antibodies. Another approach aims to optimize antibodies by improving their effector functions. While direct effects are mediated by the variable antigen binding region of the antibody, indirect effects are mediated by the constant Fc region. Efforts to improve effector function have focused primarily on regulating the Fc region. In addition, there is a need to improve the serum half-life of therapeutic antibodies, which can reduce the amount of antibody required and can increase its convenience to patients by extending the treatment interval.

[0004] For therapeutic applications, immunoglobulin G (IgG) has become the preferred choice for a number of reasons; IgG is easy to purify, relatively stable in storage, can be administered intravenously, has an extended biological half-life in vivo, and is able to exert a range of biological effector functions such as activation of complement-dependent cytotoxicity (CDC) and recruitment of effector cells by means of various Fc receptor interactions (antibody-dependent cellular cytotoxicity; ADCC). Of the five immunoglobulin classes, IgG displays the longest biological half-life due to its unique interaction with the IgG circulating receptor, the neonatal Fc receptor (FcRn). One of the known functions of this receptor is to rescue IgG from catalytic degradation. The solved FcRn-Fc co-crystal structure shows that the interaction with Fc occurs at the IgG hinge-C H 2-C H Zone 3. In the endosome, this interaction occurs at an acidic pH of 6.0-6.5 in a strictly pH-dependent manner. The bound IgG molecules are recycled back to the cell surface and released into the circulation at a physiological pH of 7.4, while the uncomplexed IgG molecules are destined for lysosomal degradation. This recycling is a mechanism that prolongs the half-life of IgG. Therefore, modulation of the FcRn-IgG interaction would allow specific control of the serum half-life of gamma immunoglobulins and Fc-fusion proteins.

[0005] Depending on the application, it may be desirable to increase or decrease the serum residence time of IgG. For therapeutic applications, a longer half-life is desired because smaller doses and fewer injections will be required. Several approaches to increase half-life have been investigated, including the use of polyethylene glycol (PEG), the generation of albumin fusion proteins or Fc fusion proteins, and the enhancement of FcRn-IgG interactions. PEGylated drugs have been used in the clinic since 1990, and PEGylation is a well-established technology for extending the residence time of drugs in the blood. Since human serum albumin (HSA) can also be recycled by FcRn in a pH-dependent interaction, some albumin fusion proteins have also been produced to enhance stability and half-life. In addition, antibody fragments fused to albumin or albumin binding domains have been shown in preclinical studies to have extended serum residence times. The generation of Fc fusion proteins is another strategy that can confer properties similar to those of intact antibodies to proteins or peptides.

[0006] The modifications of the Fc region that have been studied are summarized in Saxena (2016) Frontiers in Immunology, Volume 7, Article 580.

[0007] There remains a continuing need for antibodies with improved effector function and / or pharmacokinetics. Summary of the invention

[0008] The inventors of the present application have found that certain specific mutations in the Fc region of the antibody significantly increase the affinity of the antibody for FcRn at pH 6, while the affinity is still low at pH 7.4. Antibodies with the mutations are expected to have improved pharmacokinetic properties. In addition, the antibody exhibits excellent effector functions compared to known antibodies such as infliximab (IFX).

[0009] Therefore, the present invention relates to the subject matter defined in the following items [1] to

[88] :

[0010] [1] An antibody comprising a TNFα binding domain and an FcRn binding site, which has a high affinity for human FcRn at pH 6, wherein the high affinity is characterized by a dissociation equilibrium constant (K D ) is less than 100 nM, and further has a low affinity for human FcRn at pH 7.4, the low affinity being characterized by a K D Greater than 1 μM, wherein the amino acid sequence of the antibody comprises amino acid 434W.

[0011] [2] The antibody according to item [1], wherein the amino acid sequence of the antibody further comprises amino acid 428E and / or amino acid 311R.

[0012] [3] The antibody according to item [1] or [2], wherein the amino acid sequence of the antibody comprises amino acids 311R, 428E and 434W.

[0013] [4] An antibody according to any of the preceding items, wherein the antibody is obtained by replacing asparagine at position 434 with tryptophan (N434W), optionally replacing methionine at position 428 with glutamic acid (M428E), and optionally replacing glutamine at position 311 with arginine (Q311R).

[0014] [5] The antibody according to any of the preceding items, wherein the antibody is obtained by replacing methionine at position 428 with glutamic acid (M428E).

[0015] [6] The antibody according to any of the preceding items, wherein the antibody is obtained by replacing glutamine at position 311 with arginine (Q311R).

[0016] [7] The antibody according to any of the preceding items, comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 13.

[0017] [8] The antibody according to any of the preceding items, which has a greater affinity for human FcRn at pH 6 than infliximab.

[0018] [9] The antibody according to any of the preceding items, wherein the high affinity for human FcRn at pH 6 is characterized by a dissociation constant K D Less than 50nM.

[0019]

[10] The antibody according to any of the preceding items, wherein the high affinity for human FcRn at pH 6 is characterized by a dissociation constant K D Less than 25nM.

[0020]

[11] The antibody according to any of the preceding items, wherein the high affinity for human FcRn at pH 6 is characterized by a dissociation constant K D Less than 10nM.

[0021]

[12] An antibody according to any of the preceding items, which has affinity for human FcRn at pH 6, characterized by a dissociation constant K D In the range of 1 nM to 500 nM, or 2 nM to 100 nM, or from 3 nM to 50 nM, or 4 nM to 25 nM, or 5 nM to 10 nM.

[0022]

[13] An antibody according to any of the preceding items, wherein the high affinity or the K DDetermined by surface plasmon resonance (SPR).

[0023]

[14] The antibody according to any of the preceding items, wherein the low affinity for human FcRn at pH 7.4 is characterized by a K D Greater than 10μM.

[0024]

[15] The antibody according to any of the preceding items, wherein the K value characterizing the affinity for human FcRn at pH 7.4 is D Determined by SPR.

[0025]

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

[15] , wherein the affinity for human FcRn at pH 7.4 is so low that the K cannot be determined by SPR. D The level of value.

[0026]

[17] An antibody according to any of the preceding items, which has a K of less than 200 pM D Binds human TNFα.

[0027]

[18] An antibody according to any of the preceding items, which has a K of less than 100 pM D Binds to human TNFα.

[0028]

[19] An antibody according to any of the preceding items, which has a K of less than 50 pM D Binds human TNFα.

[0029]

[20] An antibody according to any of the preceding items, which has a K of less than 25 pM D Binds human TNFα.

[0030]

[21] An antibody according to any of the preceding items, which has a K of less than 10 pM D Binds human TNFα.

[0031]

[22] The antibody according to any of the preceding items, which is transported across a polarized cell monolayer from the apical side to the basolateral side.

[0032]

[23] An antibody according to any of the preceding items, which is transported across a polarized cell monolayer from the apical side to the basolateral side in a greater amount than a control antibody 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: 2.

[0033]

[24] The antibody according to any of the preceding items, which is transported across a polarized cell monolayer from the apical side to the basolateral side in a greater amount than infliximab.

[0034]

[25] The antibody according to item

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

[0035]

[26] The antibody according to any one of items

[23] to

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

[0036]

[27] The antibody according to any one of items

[22] to

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

[0037]

[28] An antibody according to any of the preceding items, wherein in the presence of a ten-fold excess of competing immunoglobulin, a greater percentage of the antibody is transported across a polarized cell monolayer from the apical side to the basolateral side than infliximab, wherein the percentage refers to the total mass of immunoglobulin transported across the polarized cell monolayer.

[0038]

[29] The antibody according to item

[28] , wherein the percentage of the antibody transported across polarized cell monolayers is greater than three times the percentage of the parent immunoglobulin transported across polarized cell monolayers, wherein the parent immunoglobulin differs from the antibody only in that its Fc region has only wild-type amino acids.

[0039]

[30] The antibody according to item

[28] or

[29] , wherein the percentage of antibody transported across polarized cell monolayers is greater than three times the percentage of infliximab transported across polarized cell monolayers.

[0040]

[31] The antibody according to any one of items

[22] to

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

[0041]

[32] An antibody according to any of the preceding items, which has a K of less than 100 nM, preferably less than 10 nM D Binds to CD64.

[0042]

[33] An antibody according to any of the preceding items, which has a K of less than 10 μM D Binds to CD32a(H).

[0043]

[34] An antibody according to any of the preceding items, which has a K of less than 10 μM D Binds to CD32a(R).

[0044]

[35] An antibody according to any of the preceding items, which has a K of less than 10 μM DBinds to CD32b.

[0045]

[36] An antibody according to any of the preceding items, which has a K of less than 500 nM, preferably less than 100 nM. D Binds to CD16a(V).

[0046]

[37] An antibody according to any of the preceding items, which has a K of less than 10 μM, preferably less than 1 μM D Binds to CD16a (F).

[0047]

[38] An antibody according to any of the preceding items, which has a K of less than 10 μM, preferably less than 1 μM D Binds to CD16b(NA2).

[0048]

[39] The antibody according to any of the preceding items, which binds to human C1q with a higher strength than infliximab.

[0049]

[40] An antibody according to any of the preceding items, which has a relative EC 50 and / or relative maximal mortality, with greater complement-dependent cytotoxicity of rabbit complement than infliximab.

[0050]

[41] The antibody according to any of the preceding items, which is capable of inducing CD14+CD206+ macrophages at a level equal to or greater than that of infliximab.

[0051]

[42] The antibody according to any of the preceding items, which is capable of inhibiting T cell proliferation to an extent equal to or greater than infliximab.

[0052]

[43] The antibody according to any of the preceding items, which is a non-fucosylated antibody or an antibody with reduced fucosylation.

[0053]

[44] The antibody according to any of the preceding items, comprising (i) V L A region comprising a CDR1 region having an amino acid sequence as shown in SEQ ID NO: 3, a CDR2 region having an amino acid sequence as shown in SEQ ID NO: 4, a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 5, and (ii) V H A region comprising a CDR1 region having the amino acid sequence shown in SEQ ID NO: 6, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 7, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 8.

[0054]

[45] The antibody according to any of the preceding items, which comprises a V having an amino acid sequence as shown in SEQ ID NO: 9H region and a V region having an amino acid sequence shown in SEQ ID NO: 10 L district.

[0055]

[46] The antibody according to any of the preceding items, which comprises 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.

[0056]

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

[43] , wherein the antibody comprises (i) V L A region comprising a CDR1 region having an amino acid sequence as shown in SEQ ID NO: 14, a CDR2 region having an amino acid sequence as shown in SEQ ID NO: 15, a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 16, and (ii) V H A region comprising a CDR1 region having the amino acid sequence shown in SEQ ID NO: 17, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 18, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 19.

[0057]

[48] ​​The antibody according to item

[47] , comprising a V having an amino acid sequence as shown in SEQ ID NO: 20 H A region and a V region having an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22 L district.

[0058]

[49] The antibody according to item

[47] or

[48] , which comprises 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.

[0059]

[50] The antibody according to any of the preceding items, wherein the antibody specifically binds to human TNFα.

[0060]

[51] The antibody according to any of the preceding items, wherein the antibody does not significantly bind to TNFβ.

[0061]

[52] The antibody according to any of the preceding items, wherein the antibody

[0062] (i) with a dissociation constant (K) of less than 125 pM D ) binds to human TNFα;

[0063] (ii) cross-reacts with rhesus monkey ( Macaca mulatta ) TNFα and cynomolgus monkey ( Macaca fascicularis ) TNFα;

[0064] (iii) has a potency greater than infliximab as determined by the L929 assay; and / or

[0065] (iv) capable of being synthesized in a stoichiometric ratio of at least 2 (antibody:TNFα 三聚体 ) binds to human TNFα 三聚体 .

[0066]

[53] An antibody according to any of the preceding items, which has a K of less than 1 nM D Binds TNFα from rhesus monkey.

[0067]

[54] An antibody according to any of the preceding items, which has a K of less than 1 nM D Binds TNFα from cynomolgus monkey.

[0068]

[55] The antibody according to any of the preceding items, wherein the potency of the antibody in inhibiting TNFα-induced apoptosis relative to infliximab as determined in the L929 assay is greater than 3.5, and wherein the relative potency is the IC of infliximab in the L929 assay. 50 Values ​​(in ng / mL) are compared to the IC values ​​of the antibodies described in the L929 assay. 50 The ratio of the values ​​(in ng / mL).

[0069]

[56] The antibody according to any of the preceding items, wherein the melting temperature of the variable domain of the antibody in scFv format as determined by differential scanning fluorimetry is at least 65°C.

[0070]

[57] The antibody according to any of the preceding items, wherein the melting temperature of the variable domain of the antibody in scFv format as determined by differential scanning fluorimetry is at least 68°C.

[0071]

[58] The antibody according to any of the preceding items, wherein the melting temperature as measured by differential scanning fluorimetry is at least 70°C.

[0072]

[59] The antibody according to any of the preceding items, wherein the antibody is capable of blocking the interaction between human TNFα and TNF receptor I (TNFRI).

[0073]

[60] The antibody according to any of the preceding items, wherein the antibody is capable of blocking the interaction between human TNFα and TNF receptor II (TNFRII).

[0074]

[61] The antibody according to any one of the preceding items, which is capable of inhibiting cell proliferation of peripheral blood mononuclear cells in a mixed lymphocyte reaction.

[0075]

[62] An antibody according to any of the preceding items, which is capable of inhibiting LPS-induced deregulation of CD14 + Monocytes secrete interleukin-1β.

[0076]

[63] The antibody according to item

[62] , wherein the IC inhibits LPS-induced interleukin-1β secretion 50 The value is less than 1 nM.

[0077]

[64] The antibody according to item

[63] , wherein the IC of the inhibitory effect on LPS-induced interleukin-1β secretion is 50 The value was lower than that of adalimumab.

[0078]

[65] An antibody according to any of the preceding items, which is capable of inhibiting LPS-induced deregulation of CD14 + Monocytes secrete TNFα.

[0079]

[66] The antibody according to item

[65] , wherein the IC inhibiting LPS-induced TNFα secretion 50 The value is less than 1 nM.

[0080]

[67] The antibody according to item

[66] , wherein the IC of the inhibition of LPS-induced TNFα secretion is 50 The value was lower than that of adalimumab.

[0081]

[68] The antibody according to any of the preceding items, which is immunoglobulin G (IgG), preferably IgG1.

[0082]

[69] A nucleic acid encoding the antibody of any of the preceding items.

[0083]

[70] A vector or plasmid comprising the nucleic acid of item

[69] .

[0084]

[71] A cell comprising the nucleic acid according to item

[69] or the vector or plasmid according to item

[70] .

[0085]

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

[68] , comprising culturing the cell of item

[71] in a culture medium under conditions permitting expression of a nucleic acid encoding the antibody, and recovering the antibody from the cell or from the culture medium.

[0086]

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

[68] , for use in treating inflammatory diseases or TNFα-related diseases.

[0087]

[74] The antibody for use according to item

[73] , wherein the inflammatory disease is selected from the list of disorders and diseases listed in the "Diseases to be treated" section below.

[0088]

[75] The antibody for use according to item

[73] , wherein the inflammatory disease is a gastrointestinal inflammatory disease.

[0089]

[76] The antibody for use according to item

[75] , wherein the gastrointestinal inflammatory disease is inflammatory bowel disease.

[0090]

[77] The antibody for use according to item

[75] or

[76] , wherein the gastrointestinal inflammatory disease is Crohn's disease.

[0091]

[78] The antibody for use according to item

[77] , wherein the Crohn's disease is selected from ileum, colon, ileocolonic and / or isolated upper Crohn's disease (gastric, duodenal and / or jejunal), including non-restrictive / non-penetrating, stricture, penetrating and perianal disease behaviors, allowing any combination of localizations and disease behaviors of any of the above.

[0092]

[79] The antibody for use according to item

[75] or

[76] , wherein the gastrointestinal inflammatory disease is ulcerative colitis.

[0093]

[80] The antibody for use according to item

[79] , wherein the ulcerative colitis is selected from ulcerative proctitis, enterosigmoiditis, left-sided colitis, panulo-ulcerative colitis and pouchitis.

[0094]

[81] The antibody for use according to item

[75] or

[76] , wherein the gastrointestinal inflammatory disease is microscopic colitis.

[0095]

[82] The antibody for use according to item

[73] , wherein the inflammatory disease is arthritis.

[0096]

[83] The antibody for use according to item

[73] or

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

[0097]

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

[73] to

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

[0098]

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

[73] to

[84] , wherein the method comprises topically administering the antibody.

[0099]

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

[68] .

[0100]

[87] A method for improving the transcytosis of an antibody against TNFα, comprising introducing substitutions Q311R, M428E and N434W into the amino acid sequence of the antibody.

[0101]

[88] A method for extending the plasma half-life of an antibody against TNFα, comprising introducing substitutions Q311R, M428E and N434W into the amino acid sequence of the antibody.

[0102] The present invention also relates to the subject matter defined in the following items [1] to

[18] :

[0103] [1]. An antibody comprising a TNFα binding domain and an FcRn binding site, having a high affinity for human FcRn at pH 6, wherein the high affinity is characterized by a dissociation equilibrium constant (K D ) is less than 100 nM, and further has no affinity or low affinity for human FcRn at pH 7.4, wherein the low affinity is characterized by a K D Greater than 10 μM, wherein the amino acid sequence of the antibody comprises amino acid 434W.

[0104] [2] The antibody according to item [1], wherein the amino acid sequence of the antibody further comprises amino acid 428E and / or amino acid 311R.

[0105] [3] The antibody according to item [1], wherein the amino acid sequence of the antibody comprises amino acids 311R, 428E and 434W.

[0106] [4] The antibody according to any one of the preceding items, which has an affinity for human FcRn at pH 6 that is greater than the affinity of infliximab for human FcRn at pH 6.

[0107] [5]. The antibody according to any one of the preceding items, wherein the high affinity for human FcRn at pH 6 is characterized by a dissociation constant K D Less than 10nM.

[0108] [6] An antibody according to any one of the preceding items, which has a K of less than 100 pM D Binds human TNFα.

[0109] [7] The antibody according to any one of the preceding items, which is transported across the polarized cell monolayer from the apical side to the basolateral side in a greater amount than infliximab.

[0110] [8] The antibody according to item [7], wherein the amount of the antibody transported across the polarized cell monolayer is greater than twice the amount of infliximab transported across the polarized cell monolayer.

[0111] [9]. An antibody according to any of the preceding items, wherein in the presence of a ten-fold excess of competing immunoglobulin, a greater percentage of the antibody is transported across a polarized cell monolayer from the apical side to the basolateral side than infliximab, wherein the percentage refers to the total mass of immunoglobulin transported across the polarized cell monolayer.

[0112]

[10] The antibody according to item [9], wherein the percentage of antibody transported across the polarized cell monolayer is greater than three times the percentage of infliximab transported across the polarized cell monolayer.

[0113]

[11] . The antibody according to any one of the preceding items, which is a non-fucosylated antibody or an antibody with reduced fucosylation.

[0114]

[12] The antibody according to any one of the preceding items, for use in treating inflammatory diseases.

[0115]

[13] The antibody for use according to item

[12] , wherein the inflammatory disease is inflammation of the gastrointestinal tract.

[0116]

[14] The antibody for use according to item

[12] or

[13] , wherein the treatment comprises oral administration of an effective amount of the antibody.

[0117]

[15] The antibody for use according to item

[12] or

[13] , wherein the antibody is administered topically.

[0118]

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

[11] .

[0119]

[17] . A method for improving the transcytosis of an antibody against TNFα, comprising introducing substitutions Q311R, M428E and N434W into the amino acid sequence of the antibody.

[0120]

[18] . A method for prolonging the plasma half-life of an antibody against TNFα, comprising introducing substitutions Q311R, M428E and N434W into the amino acid sequence of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 : Potency of anti-TNFα antibody variants in neutralizing human TNFα in the L929 assay. Dose response curves for anti-TNFα antibody variants and the reference infliximab are shown.

[0122] Figure 2: Transport of anti-TNFα IgG variants across polarized T84 cells. The amount of anti-TNFα antibody variants and infliximab (IFX) from the apical to the basolateral reservoir 4 hours after addition is shown. Expressed as ng / cm 2 Error bars represent SD of two to four independent monolayers.

[0123] Figure 3 : Transport of anti-TNFα IgG variants across polarized T84 cells in the presence of excess myeloma IgG. Shown is the amount of anti-TNFα Ab variants and IFX 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 / cm 2 Error bars represent SD of three to four independent monolayers.

[0124] Figure 4 : ADCC activity. ADCC induction by anti-TNFα antibody variants, wild-type antibody and IFX.

[0125] Figure 5 : Binding to human C1q. Binding of IFX and anti-TNFα antibody variants to human C1q. Each concentration was determined in duplicate. Error bars represent SD.

[0126] Figure 6 : CDC activity. Percentage of cell death for various anti-TNFα variants compared to IFX. Each sample point is the average of 6 independent replicates.

[0127] Figure 7 : The effect of each compound on CD14 relative to the induction of IFX + CD206 + Induction of macrophages. Pooled data from 4 independent experiments. Columns represent mean values ​​and error bars represent SEM.

[0128] Figure 8 : Each compound inhibits T cell proliferation relative to IFX. Pooled data from 3 independent experiments. Bars represent mean values ​​and error bars represent SEM.

[0129] Fig. 9 : Schematic representation of site-directed mutagenesis.

[0130] Fig.10 : Schematic representation of the major N-glycan forms attached to N297 of anti-TNFα antibody variants. The two major N-glycan profiles in the tested anti-TNFα antibody variants were 4GlcNac-1Fuc-3Man and 4GlcNac-1Fuc-3Man-1Gal, while for IgG variants produced in the presence of 2FF, the same biantennary structure occurred except for the lack of fucose. DETAILED DESCRIPTION

[0131] The present invention relates to antibodies that can bind to TNFα and comprise an FcRn binding site. According to the present application, if the antibody is able to bind to FcRn at pH 6, preferably to human FcRn, then it comprises an FcRn binding site. Binding to FcRn at pH 6 can be determined by SPR, for example as described in Example 4 of the present application. If the binding of the antibody to FcRn at pH 6 can be detected by SPR, then the antibody has an FcRn binding site. The antibody of the present invention has a high affinity for human FcRn at pH 6, characterized by a dissociation equilibrium constant (K D ) is less than 100 nM. The antibody has low affinity for human FcRn at pH 7.4, characterized in that K D Greater than 1 μM. The amino acid sequence of the antibody comprises the amino acid tryptophan at position 434 (EU numbering).

[0132] Throughout the specification and claims, 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 (Kabat et al., Sequences of Immunological Interest. 5th Edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) is generally used. When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) as reported in the EU index, expressly incorporated herein by reference). Unless otherwise indicated herein, reference to residue numbers in the variable domain of an antibody refers to residue numbering by the Kabat numbering system. Unless otherwise indicated herein, references to residue numbers in an antibody constant domain refer to residue numbering by the EU numbering system (see, e.g., WO 2006 / 073941).

[0133] Antibody

[0134] In the context of the present application, the term "antibody" is used as a synonym for "immunoglobulin" (Ig), which is defined as a protein belonging to the IgG, IgM, IgE, IgA or IgD class (or any subclass thereof), and includes all conventionally known antibodies and functional fragments thereof. In the context of 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 substantially retains one or more characteristics of these parent antibodies. An "antigen-binding fragment" or "antigen-binding domain" of an antibody / immunoglobulin is defined as a fragment (e.g., a variable region of IgG) that retains an antigen-binding region. An "antigen-binding region" of an antibody is typically found in one or more hypervariable regions of an antibody, i.e., CDR-1, CDR-2 and / or CDR-3 regions. The antibody of the present invention may be part of a bifunctional or multifunctional construct.

[0135] Preferably, the antibody is a monoclonal antibody. The term "monoclonal antibody" as used herein 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, rather than its production method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display technologies or combinations thereof. (Harlow and Lane, "Antibodies, A Laboratory Manual" CSH Press 1988, Cold Spring Harbor NY).

[0136] In other embodiments, including embodiments involving the use of anti-TNFα antibodies in humans, chimeric, primate, humanized or human antibodies may be used. In a preferred embodiment, the antibody is a human antibody or a humanized antibody, more preferably a monoclonal human antibody or a monoclonal humanized antibody. In another specific embodiment, the antibody of the present invention is an immunoglobulin, preferably immunoglobulin G (IgG). The subclass of IgG of the present invention is not limited, including IgG1, IgG2, IgG3 and IgG4. Preferably, the IgG of the present invention is subclass 1, 2 or 4, i.e., it is an IgG1, IgG2 or IgG4 molecule, respectively. Most preferably, the IgG of the present invention is subclass 1, i.e., it is an IgG1 molecule.

[0137] TNFα binding domain

[0138] 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.

[0139] Preferably, the antibodies of the present invention specifically bind to TNFα. As used herein, an antibody "specifically recognizes" or "specifically binds" to human TNFα when the antibody is able to distinguish between human TNFα and one or more reference molecules. Preferably, the IC binding to each reference molecule is 50 IC value of binding to TNFα 50 The value is at least 1000 times greater. In its most general form (and when the reference for the definition is not mentioned), "specific binding" refers to the ability of an antibody to distinguish between human TNF α and unrelated biomolecules, such as determined by specific determination methods known in the art. Such methods include, but are not limited to, Western blot and ELISA tests. For example, standard ELISA assays can be performed. Usually, the determination of binding specificity is not by using a single reference biomolecule, but by using a group of about three to five unrelated biomolecules, such as milk powder, BSA, transferrin, etc. In one embodiment, specific binding refers to the ability of an antibody to distinguish between human TNF α and human TNF β.

[0140] The antibody of the present invention comprises V L Domain and V H Domain. V L The structural domain includes CDR1 region (CDRL1), CDR2 region (CDRL2), CDR3 region (CDRL3) and framework region. H The domains include the CDR1 region (CDRH1), the CDR2 region (CDRH2), the CDR3 region (CDRH3) and the framework region.

[0141] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody, which primarily contributes to antigen binding. A most commonly used definition of these six CDRs can be found in Kabat EA et al. (1991) Sequences of proteins of immunological interest, NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2 and CDR3 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), and CDR2 and CDR3 of the heavy chain. Chain variable domain (CDR H2, CDR H3 or H2, H3). However, the CDR1 (CDR H1 or H1) of the heavy chain variable domain used herein is defined by the following residues (Kabat numbering): starting at position 26 and ending at position 36.

[0142] In one embodiment of the invention, the antibody of the invention is an anti-TNFα antibody as originally filed as disclosed in any one of PCT applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 and PCT / EP2017 / 056227. In 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 a complementarity determining region (CDR) having an amino acid sequence as originally disclosed in PCT applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 and PCT / EP2017 / 056227.

[0143] In a preferred embodiment of the present 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 an amino acid sequence as disclosed in PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 or PCT / EP2017 / 056227. In another preferred embodiment of the present invention, the antibody is an anti-TNFα antibody having a light chain variable domain and a heavy chain variable domain comprising CDRs having an amino acid sequence as disclosed in claim 2 of originally filed PCT / EP2017 / 056218, claim 2 of PCT / EP2017 / 056246, claim 2 of PCT / EP2017 / 056237 or claim 2 of PCT / EP2017 / 056227. In another preferred embodiment of the present invention, the anti-TNFα antibody is selected from an anti-TNFα antibody comprising a heavy chain variable domain amino acid sequence and / or a 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 disclosure of each of the international patent applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 and PCT / EP2017 / 056227 is incorporated herein in its entirety. They constitute part of the disclosure of the present application.

[0144] In a specific embodiment, the antibody of the invention comprises (i) V LA region comprising a CDR1 region having an amino acid sequence as shown in SEQ ID NO: 3, a CDR2 region having an amino acid sequence as shown in SEQ ID NO: 4, a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 5, and (ii) V H A region comprising a CDR1 region having the amino acid sequence shown in SEQ ID NO: 6, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 7, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 8.

[0145] In a more preferred embodiment, the antibody of the present invention comprises a V having an amino acid sequence as shown in SEQ ID NO: 9. H In another more preferred embodiment, the antibody comprises a V region having an amino acid sequence as shown in SEQ ID NO: 10. L Most preferably, the antibody of the present invention comprises (i) a V region having an amino acid sequence as shown in SEQ ID NO: 9 H region, and (ii) a V region having an amino acid sequence as shown in SEQ ID NO: 10 L district.

[0146] In another specific embodiment, the antibody of the present invention comprises (i) V L A region comprising a CDR1 region having an amino acid sequence as shown in SEQ ID NO: 14, a CDR2 region having an amino acid sequence as shown in SEQ ID NO: 15, a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 16, and (ii) V H A region comprising a CDR1 region having the amino acid sequence shown in SEQ ID NO: 17, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 18, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 19.

[0147] In a more preferred embodiment, the antibody of the present invention comprises a V having an amino acid sequence as shown in SEQ ID NO: 20. H In another more preferred embodiment, the antibody comprises a V region having an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22. L Most preferably, the antibody of the present invention comprises (i) a V region having an amino acid sequence as shown in SEQ ID NO: 20 H region, and (ii) a V region having an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22 L district.

[0148] The antibodies of the present invention have a high affinity for human TNFα. D ” refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, antibodies of the invention dissociate at a constant constant of less than about 2×10 -10 M, preferably less than 1.5×10 -10 M, preferably less than 1.25×10 -10 M, more preferably less than 1×10 -10 M, most preferably less than 7.5×10 -11 M or even less than 5×10 -11 The dissociation equilibrium constant (K D ) in combination with human TNFα. In particular, K D Determination of.

[0149] Modifications that affect affinity for FcRn

[0150] 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 to human FcRn. Typically, at pH 6, at least one amino acid modification increases the affinity of the antibody to human FcRn. In one embodiment, the at least one amino acid modification increases the affinity of the antibody to human FcRn at pH 6, wherein it does not substantially change the affinity to human FcRn at pH 7.4. Preferably, the modified antibody has at least one amino acid substitution, such as 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 a wild-type antibody or a parent antibody. Preferably, 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 modifications may affect the binding of FcRn, such as by structural changes. Amino acid modifications can be produced by known methods per se, for example by site-directed mutagenesis as described in "Antibody Engineering—Methods and Protocols," edited by Patrick Chames, 2012, Chapter 31 (ISBN 978-1-61779-973-0).

[0151] The antibodies of the present invention comprise the amino acid tryptophan at position 434 (EU numbering). It is referred to herein as "434W". The natural amino acid at position 434 of unmodified human IgG antibodies is asparagine (N). Therefore, the antibodies of the present invention can be obtained by introducing the mutation N434W into the antibody. Preferably, the antibodies of the present invention can be obtained or obtained by replacing asparagine with tryptophan at position 434.

[0152] The antibodies of the present invention comprise the amino acid glutamic acid at position 428 (EU numbering). It is referred to herein as "428E". The natural amino acid at position 428 of unmodified human IgG antibodies is methionine (M). Therefore, the antibodies of the present invention can be obtained by introducing the mutation M428E into the antibody. Preferably, the antibodies of the present invention can be obtained or obtained by replacing methionine with glutamic acid at position 428.

[0153] Preferably, the antibody of the present invention further comprises the amino acid arginine at position 311 (EU numbering). It is referred to herein as "311R". The natural amino acid at position 311 of the unmodified human IgG antibody is glutamine (Q). Therefore, the antibody of the present invention can be obtained by introducing the mutation Q311R into the antibody. Preferably, the antibody of the present invention can be obtained or obtained by replacing arginine with glutamine at position 311.

[0154] In one embodiment, the antibody of the present invention comprises amino acid 434W and amino acid 428E. In another embodiment, the antibody of the present invention comprises amino acid 434W and amino acid 311R. In a preferred embodiment of the present invention, the antibody comprises amino acids 434W, 428E and 311R. The antibody can be obtained by introducing Q311R, M428E and N434W mutations into the antibody.

[0155] The remaining amino acid sequence of the constant domain may be identical to the natural amino acid sequence of a typical human IgG. However, the amino acid sequence of the antibody may contain one or more additional mutations or substitutions to the natural amino acid sequence of the Fc region of a native antibody, as long as the antibody still has TNFα binding activity and effector function.

[0156] In a preferred embodiment, the Fc region of the antibody of the present invention, including the hinge region, comprises or consists of the amino acid sequence shown in SEQ ID NO:13.

[0157] In one embodiment, the heavy chain of the antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 11. Preferably, the antibody further comprises a light chain having the amino acid sequence shown in SEQ ID NO:1.

[0158] In another embodiment, the heavy chain of the antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 12. Preferably, the antibody further comprises a light chain having the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24.

[0159] In a preferred aspect of the present invention, the antibody of the present invention is a non-fucosylated antibody or an antibody with reduced fucosylation.

[0160] 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 determining the percentage of fucosylation are known in the art. Preferably, the percentage of fucosylation is determined as described in Example 11 of the present application.

[0161] 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 15% of the N-glycans of the antibody are fucosylated. In a specific embodiment, the N-glycans of the antibodies of the invention do not contain any fucose.

[0162] Preferably, less than 90% of the N-glycans of 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 of N297 (EU numbering) of the antibody are fucosylated. Most preferably, less than 15% of the N-glycans of N297 (EU numbering) of the antibody are fucosylated.

[0163] In another embodiment, the N-glycan at N297 of the antibody does not comprise any fucose.

[0164] Non-fucosylated antibodies, sometimes also referred to as afucosylated antibodies, can be produced by various methods. For example, coordinated knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) genes in CHO cells can be used to generate monoclonal antibody variants that are completely afucosylated and have enhanced ADCC (see, e.g., Imai-Nishiya et al. (2007) BMC Biotechnol. 7, 84). A method using zinc finger nucleases (ZFNs) to cleave the FUT8 gene in the region encoding the catalytic core of α1,6-fucosyltransferase and disrupt the corresponding enzyme function in CHO cells can be used to produce monoclonal antibodies that completely lack core fucose (see, e.g., Malphettes et al. (2010) Biotechnol. Bioeng. 106, 774-783).

[0165] Antibodies with reduced fucosylation can be prepared by adding a decoy substrate such as 2-deoxy-2-fluoro-2-fucose to the culture medium (see, e.g., Dekker et al. (2016) Sci Rep 6:36964), resulting in reduced incorporation of fucose into IgG-Fc glycans.

[0166] In another embodiment, the antibodies of the invention have 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).

[0167] Affinity for FcRn

[0168] The antibodies of the present invention have a high affinity for human FcRn at pH 6. The high affinity binding of the antibodies to human FcRn at pH 6 is characterized by a K D Preferably, the K for high affinity binding at pH 6 is less than 100 nM. D The value is less than 75 nM, or less than 50 nM, or less than 25 nM, or even less than 10 nM. For example, the K value characterizing the affinity for human FcRn at pH 6 is D Values ​​may range from 1 nM to 500 nM, or from 2 nM to 100 nM, or from 3 nM to 50 nM, or from 4 nM to 25 nM, or from 5 nM to 10 nM.

[0169] In a preferred embodiment, the affinity of an antibody of the invention for human FcRn at pH 6 is greater than the affinity of infliximab for human FcRn at pH 6.0.

[0170] The affinity of the antibodies of the invention for human FcRn is preferably determined by surface plasmon resonance (SPR), for example, as described in Example 4 of the present application.

[0171] The antibodies of the present invention generally have low affinity for human FcRn at pH 7.4. The low affinity is characterized by a K D Preferably, the low affinity for human FcRn at pH 7.4 is characterized by a K D The value is greater than 2 μM, or greater than 5 μM, or greater than 10 μM.

[0172] In certain embodiments, low affinity is so low that the K cannot be determined by SPR. D The level of value.

[0173] In a specific embodiment, (i) the K of the binding of an antibody of the invention to human FcRn at pH 7.4 is D (ii) K value of the antibody of the present invention binding to human FcRn at pH 6.0 D The ratio of the values ​​is at least 200, or at least 300, or at least 400, or at least 500, or at least 600, or at least 700, or at least 800, or at least 900, or at least 1000.

[0174] Functional properties of antibodies

[0175] The antibodies of the present invention are effectively transported from the apical side across the polarized cell monolayer to the basolateral side. Typically, the amount of transport across the polarized cell monolayer is greater than the amount of infliximab, wherein the amount of antibody or infliximab refers to the mass of the polarized cell monolayer per square centimeter. 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 125%, more preferably at least 150% or at least 175%, or at least 200% (wherein the transport amount of infliximab is set to 100%).

[0176] Furthermore, in the presence of excess competing immunoglobulin, antibodies are specifically transported across polarized cell monolayers from the apical side to the basolateral side. This is referred to herein as specific transport.

[0177] In the presence of a 10-fold excess of competing immunoglobulin, the percentage of the total mass of immunoglobulin transported across the polarized cell monolayer from the apical side to the basolateral side is greater than the percentage of infliximab transported across the polarized cell monolayer. The percentage of the 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 150%, or at least 200%, or at least 250%, or at least 300% relative to the percentage of infliximab transported across the polarized cell monolayer in the presence of a 10-fold excess of an irrelevant immunoglobulin (with infliximab set at 100%).

[0178] Preferably, the polarized cell monolayer is a monolayer of polarized T84 cells.The transport assay mimicking transcytosis can be performed as described in Example 5 of the present application.

[0179] The antibodies of the present invention bind to CD64, CD32a(H), CD32a(R), CD32b, CD16a(V), CD16a(F) and CD16b(NA2).

[0180] The antibodies of the present invention generally have a K of less than 100 nM, preferably less than 10 nM. D Binds to CD64.

[0181] The antibodies of the present invention generally have a K of less than 10 μM. D Binding to CD32a (H).

[0182] The antibodies of the present invention generally have a K of less than 10 μM. D Binds to CD32a.

[0183] The antibodies of the present invention generally have a K of less than 10 μM. D Binds to CD32b.

[0184] The antibodies of the present invention generally have a K of less than 500 nM, preferably less than 100 nM. D Binds to CD16a (V).

[0185] The antibodies of the present invention generally have a K of less than 10 μM, preferably less than 1 μM. D Binding to CD16a (F).

[0186] The antibodies of the present invention generally have a K of less than 10 μM, preferably less than 1 μM. D Binds to CD16b (NA2).

[0187] The antibodies of the invention further bind to human C1q. Preferably, this binding is stronger than the binding of infliximab to human C1q.

[0188] The antibody of the present invention also has the complement-dependent cytotoxicity (CDC) of rabbit complement. 50 And / or relative to maximum mortality, the CDC of the antibody of the present invention is preferably greater than the CDC of infliximab.

[0189] The antibodies of the present invention can also induce CD14 + CD206 + The induction level is preferably similar to, equal to or greater than that of infliximab.

[0190] The antibodies of the present invention are also capable of inhibiting T cell proliferation. The degree of inhibition of T cell proliferation is preferably similar to, equal to or greater than the degree of inhibition of T cell proliferation by infliximab.

[0191] Drug ingredients and treatment

[0192] Treatment of disease includes treatment of patients who have been diagnosed with any form of disease at any clinical stage or manifestation; delaying the onset, development or exacerbation or worsening of symptoms or signs of disease; and / or preventing and / or lessening the severity of disease.

[0193] 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 aspects, the human is a pediatric patient. In other aspects, the human is an adult patient.

[0194] Compositions comprising an anti-TNFα antibody and optionally one or more other therapeutic agents (e.g., a second therapeutic agent described below) are described herein. The composition is generally provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition can be in any suitable dosage form (depending on the desired method of administering it to a patient).

[0195] Anti-TNFα antibodies can be administered to patients by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intrathecal, topically (e.g., through a mucosa). In any given case, the most appropriate route of administration depends on the specific antibody, the subject, and the nature and severity of the disease and the subject's physical condition. Typically, anti-TNFα antibodies will be administered intravenously.

[0196] In a particularly preferred embodiment, the antibody of the invention is administered orally. If administered by the oral route, the antibody is preferably IgG, most preferably IgG1.

[0197] In typical embodiments, the anti-TNFα antibody is present in the pharmaceutical composition at a concentration sufficient to allow intravenous administration at 0.5 mg / kg to 20 mg / kg body weight. In some embodiments, concentrations of antibodies 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 concentrations between the foregoing values, such as 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, or 10 mg / kg to 18 mg / kg.

[0198] The effective dose of the anti-TNFα antibody per single (e.g., bolus) administration, multiple administrations, or continuous administration can be in the range of about 0.001 to about 750 mg / kg, or a serum concentration of 0.01-5000 μg / ml per single (e.g., bolus) administration, multiple administrations, 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. When taken orally, serum concentrations may be very low, even below the limit of detection. In certain embodiments, each dose may be in the range of about 0.5 mg to about 50 mg per kilogram of body weight or about 3 mg to about 30 mg per kilogram of body weight. The antibody can be formulated into an aqueous solution.

[0199] In a particularly preferred embodiment, the antibodies of the present invention are administered orally. If administered by oral route, the antibody is preferably IgG, most preferably IgG1. If the antibody is administered orally, the daily dose of the antibody is generally about 0.01 mg / kg to about 100 mg / kg body weight, or about 0.05 mg / kg to about 50 mg / kg body weight, or about 0.1 mg / kg to about 25 mg / kg body weight, or about 0.15 mg / kg to about 10 mg / kg body weight, or about 0.16 mg / kg to about 5 mg / kg body weight, or about 0.2 mg / kg to about 2 mg / kg body weight, or about 0.2 mg / kg to about 1 mg / kg body weight. Typically, a favorable dosage is 1 to 200 mg per day, preferably 5 to 100 or 10 to 50 mg per day.

[0200] The pharmaceutical composition can be conveniently presented in unit dosage form, each unit dosage containing a predetermined amount of anti-TNFα antibody. Such a unit may contain 0.5 mg to 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. The pharmaceutically acceptable carrier can take a variety of forms, for example depending on the condition to be treated or the route of administration.

[0201] Determination of an effective dose of an anti-TNFα antibody, the total number of doses, and duration of treatment is well within the capabilities of those skilled in the art and can be determined using standard dose escalation studies.

[0202] Therapeutic formulations of anti-TNFα antibodies suitable for the methods described herein can be prepared by mixing the antibody having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (all of which are referred to herein as "carriers") commonly used in the art, i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants and other various additives, for storage in the form of lyophilized formulations or aqueous solutions. See Remington's Pharmaceutical Sciences, 16th edition (Osol ed., 1980). Such additives must be nontoxic to the recipient at the dosages and concentrations employed.

[0203] Buffer helps to maintain pH in the range close to physiological conditions. They can exist with a concentration of about 2mM to about 50mM. Suitable buffers include organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), citric acid-phosphate buffers, succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-fumaric acid In some embodiments, the present invention provides the following buffers: (a mixture of disodium succinate, a mixture of fumaric acid and disodium fumarate, a mixture of monosodium fumarate and disodium fumarate, etc.), a gluconate buffer (e.g., a mixture of gluconic acid and sodium saccharate, a mixture of gluconic acid and sodium hydroxide, a mixture of gluconic acid and potassium ammonium oxalate, etc.), an oxalate buffer (e.g., a mixture of oxalic acid and sodium oxalate, a mixture of oxalic acid and sodium hydroxide, a mixture of oxalic acid and potassium oxalate, etc.), a lactic acid buffer (e.g., a mixture of lactic acid and sodium lactate, a mixture of lactic acid and sodium hydroxide, a mixture of lactic acid and potassium lactate, etc.), and an acetate buffer (e.g., a mixture of acetic acid and sodium acetate, a mixture of acetic acid and sodium hydroxide, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

[0204] The pharmaceutical composition of the present invention may further comprise at least one salt, such as sodium chloride. The concentration of the salt is preferably in the range of 100 mM to 200 mM, such as between 100-200 mM, such as about 150 mM.

[0205] Preservatives can be added to prevent microbial growth, and preservatives can be added in an amount of 0.2%-1% (w / v). Suitable preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzyl aluminum halides (e.g., chlorides, bromides, and iodides), hexamethylammonium chloride, and alkylparabens (e.g., methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes referred to as "stabilizers," can be added to ensure the isotonicity of the liquid composition, and include polyols, preferably trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a wide range of excipients, and their functional range can range from fillers to soluble therapeutic agents or additives that help prevent denaturation or adhesion to the container wall. Typical stabilizers can be polyols (listed above); amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc., including cyclotols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing Reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, o-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of less than 10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose; trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer is present in an amount of 0.1 to 10,000 weight per weight of active protein.

[0206] Nonionic surfactants or detergents (also called "wetting agents") may be added to help dissolve the therapeutic agent and protect the therapeutic protein from aggregation caused by agitation, which also exposes the formulation to stressed shear surfaces without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxalates (184, 188, etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers ( -20, -80, etc.). The nonionic surfactant may 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.

[0207] Other miscellaneous excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), protease inhibitors, and solubilizing agents.

[0208] The formulations herein may also include a second therapeutic agent in addition to the anti-TNFα antibody thereof. Examples of suitable second therapeutic agents are provided below.

[0209] The dosing schedule can range from once a month to once a day, depending on a number of clinical factors, including the type of disease, the severity of the disease, and the patient's sensitivity to the anti-TNFα antibody. In a specific embodiment, the anti-TNFα antibody is administered daily, twice a week, three times a week, every other day, every five days, once a week, every ten days, every two weeks, every three weeks, every four weeks, or once a month, or any range between any two values ​​​​above, for example, from every four days to monthly, from every 10 days to every two weeks, or twice to three times a week, etc.

[0210] The dosage of the anti-TNFα antibody administered will vary depending on the specific antibody, the subject, the nature and severity of the disease, the subject's physical condition, the treatment regimen (e.g., whether a second therapeutic agent is used), and the selected route of administration; appropriate dosages can be readily determined by those skilled in the art.

[0211] Those skilled in the art will recognize that the optimal amount and interval of individual doses of their anti-TNFα antibody will be determined by the nature and extent of the disease being treated, the form, route and site of administration, and the age and condition of the specific subject to be treated, and the physician will ultimately determine the appropriate dose to use. The dose may be repeated as appropriate. If side effects occur, the number and / or frequency of doses may be changed or reduced in accordance with normal clinical practice.

[0212] Disease to be treated

[0213] The present invention relates to a method for treating or preventing a disease associated with human TNFα in a subject, comprising administering to the subject an antibody as defined herein. The term "TNFα-associated disease" or "TNFα-associated disease" refers to a disease that requires the involvement of TNFα for the onset, development or persistence of any condition, symptom or disease state.Typical diseases associated with TNFα include, but are not limited to, general chronic and / or autoimmune inflammatory states, general immune-mediated inflammatory diseases, inflammatory CNS diseases, inflammatory diseases affecting the eyes, joints, skin, mucous membranes, central nervous system, gastrointestinal tract, urinary tract or lungs, general uveitic states, retinitis, HLA-B27+ uveitis, Behcet's disease, dry eye, glaucoma, Sjögren's syndrome, diabetes (including diabetic neuropathy), insulin resistance, general arthritic states, rheumatoid arthritis, osteoarthritis, reactive arthritis and Reiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiple sclerosis, Gram-negative Barré syndrome, myasthenia gravis, amyotrophic lateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidney disease, cystitis, psoriasis (including psoriatic arthritis), hidradenitis suppurativa, panniculitis, pyoderma gangrenosum, SAPHO syndrome (synovitis, acne, impetigo, osteoporosis, and osteitis), acne, Sweet syndrome, pemphigus, Crohn's disease (including extraintestinal manifestations), ulcerative colitis, bronchial asthma, hypersensitivity pneumonitis, general allergy, allergic rhinitis, allergic sinusitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, Wegener's granulomatosis, Kawasaki syndrome, giant cell arteritis, polysinusitis, burns, graft-versus-host disease Host disease, host graft reaction, rejection after organ or bone marrow transplantation, systemic and local vasculitis of the systemic, systemic and cutaneous lupus erythematosus, polymyositis and dermatomyositis, sclerosis, pre-eclampsia, acute and chronic pancreatitis, viral hepatitis, alcoholic hepatitis, postoperative inflammation, for example inflammation after ophthalmic surgery (e.g. cataract (lens replacement) or glaucoma surgery), joint surgery (including arthroscopic surgery), surgery on joint-related structures (e.g. ligaments), oral and oral / or dental surgery, minimally invasive cardiovascular surgery (e.g. PTCA, atherectomy, stenting, laparoscopic and / or endoscopic gynecological and medical surgery, endoscopic urological surgery (e.g. Prostate surgery, ureteroscopy, cystoscopy, interstitial cystitis) or general perioperative inflammation (prophylaxis), bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, pustular dermatitis, cerebral malaria, hemolytic uremic syndrome, allograft rejection, otitis media, snake bites, erythema nodularis, myelodysplastic syndrome, primary sclerosing cholangitis, seronegative spondylomalacia, autoimmune hemolytic anemia, orofacial granulocytosis, proliferative purulent stomatitis, aphthous stomatitis, geographic glossitis, migratory stomatitis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Bell's palsy, Creutzfeldt-Jakob disease and general neurodegenerative diseases.

[0214] Cancer-related osteolysis, cancer-related inflammation, cancer-related pain, cancer-related cachexia, bone metastases, acute and chronic forms of pain, whether these are caused by central or peripheral actions of TNFα and whether they are classified as inflammatory, nociceptive or neuropathic forms 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, menstrual disorders.

[0215] Specific diseases to be treated include arthritis in general, rheumatoid arthritis, osteoarthritis, reactive arthritis, juvenile arthritis; psoriasis, including psoriatic arthritis; inflammatory bowel disease, including Crohn's disease, ulcerative colitis, including proctitis, sigmoiditis, enterosigmoiditis, left-sided colitis, extensive colitis and pancreatitis, indeterminate colitis, microscopic colitis (including collagenous and lymphocytic colitis), connective tissue disease colitis, diverticular colitis, eosinophilic colitis and pouchitis.

[0216] Most preferably, the antibodies of the invention are used to treat inflammatory bowel disease, in particular Crohn's disease, ulcerative colitis or microscopic colitis. Crohn's disease can be ileal, colonic, ileocolic or isolated upper Crohn's disease (gastric, duodenal and / or jejunal), including non-stricturing / non-penetrating, stricture, penetrating and perianal disease behavior, allowing any combination of disease localization and disease behavior of any of the above. Ulcerative colitis can be ulcerative proctitis, proctosigmoiditis, left-sided colitis, panulcerative colitis and pouchitis.

[0217] Combination therapy and other aspects

[0218] Preferably, the patient treated with the anti-TNFα antibody is also treated with another conventional drug. For example, patients with inflammatory bowel disease, especially if they have moderate to severe disease, are often also treated with mesalazine or its derivatives or prodrugs, corticosteroids (e.g., budesonide or prednisolone (oral or intravenous)), immunosuppressants (e.g., 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, golimumab), integrin antagonists (e.g., natalizumab, vedolizumab), anti-IL-23 antibodies (e.g., MED2070), anti-β7 antibodies (e.g., edrema), JAK inhibitors in the JAK-STAT pathway (e.g., tofacitinib), and the like. To maintain stable and longer remissions, other drugs that may be co-administered to the patient include immunosuppressants such as azathioprine / 6-MP or methotrexate or oral cyclosporine. Another aspect of the invention is the use of an anti-TNFα antibody as defined above for reducing inflammation.

[0219] Another aspect of the invention is an anti-TNFα antibody as defined above for use in reducing inflammation in a patient suffering from an inflammatory disorder.

[0220] Another aspect of the invention is a method for treating an inflammatory disorder, comprising administering to a patient in need thereof an effective amount of an anti-TNFa antibody as defined above. The inflammatory disorder is preferably one of the disorders mentioned above.

[0221] Another aspect of the invention is a method for preventing an inflammatory disorder, comprising administering to a patient in need thereof an effective amount of an anti-TNFa antibody as defined above. The inflammatory disorder is preferably one of the disorders mentioned above.

[0222] Another aspect of the present invention is a method for improving the transcytosis of an antibody against TNFα, comprising introducing substitutions Q311R, M428E and N434W into the amino acid sequence of the antibody to obtain a modified antibody with improved transcytosis. The modified antibody is preferably an antibody as described above.

[0223] Another aspect of the present invention is a method for extending the plasma half-life of an antibody against TNFα, comprising introducing substitutions Q311R, M428E and N434W 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 above. The plasma half-life of the non-modified antibody (i.e., the parent antibody lacking substitutions Q311R, M428E and N434W) may be increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%.

[0224] Table 1: Sequence overview of the sequence listing

[0225] SEQ ID NO: Description of amino acid sequence 1 The light chain of the parent antibody Ab-wt of the modified antibody used in the example 2 The heavy chain of the parent antibody Ab-wt of the modified antibody used in the examples 3 CDR L1 of clone 16-22-H05 4 CDR L2 of clone 16-22-H05 5 CDR L3 of clone 16-22-H05 6 CDR H1 of clone 16-22-H05 7 CDR H2 of clone 16-22-H05 8 CDR H3 of clone 16-22-H05 9 <![CDATA[Humanized IgG V of Clone 16-22-H05 H > 10 <![CDATA[V of humanized IgG of Clone 16-22-H05 L > 11 Heavy chain of Ab-REW (based on clone 16-22-H05) 12 Heavy chain of Ab-REW (based on clone 17-22-B03) 13 Fc region of Ab-REW (including hinge region) 14 CDR L1 of clone 17-22-B03 15 CDR L2 of clone 17-22-B03 16 CDR L3 of clone 17-22-B03 17 CDR H1 of clone 17-22-B03 18 CDR H2 of clone 17-22-B03 19 CDR H3 of clone 17-22-B03 20 <![CDATA[V of humanized IgG of Clone 17-22-B03 H > 21 <![CDATA[V of humanized IgG of Clone 17-22-B03 L (sc08)]]> 22 <![CDATA[V of humanized IgG of Clone 17-22-B03 L (sc02)]]> 23 Humanized IgG light chain of clone 17-22-B03 (sc08) 24 Humanized IgG light chain of clone 17-22-B03 (sc02)

[0226] Example

[0227] Antibody variants

[0228] Several variants of anti-TNFα antibodies (hereinafter referred to as "parent antibodies" or "Ab-wt") were generated by introducing substitutions in the Fc region of the antibody amino acid sequence. The light chain of Ab-wt has the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain of Ab-wt has the amino acid sequence shown in SEQ ID NO: 2. Mutations were introduced by site-directed mutagenesis using established methods. Briefly, mutations were introduced by PCR. The forward primer was designed to contain the desired mutation, while the reverse primer was designed so that the 5' ends of the two primers annealed back to back (but not overlapping) ( Fig. 9 ). PCR was performed for 25 cycles (98°C for 10 s, 64°C for 30 s, 72°C for 3 min). Before running the PCR products on an agarose gel, the non-mutated PCR template was removed from the PCR product pool using the restriction enzyme Dpnl. After gel purification of the PCR products, the blunt ends were ligated to obtain a circularized plasmid, which was transformed into competent E. coli cells. After overnight incubation, several colonies were picked, and the plasmid DNA was isolated and sequenced to confirm that the mutation had been incorporated. Non-fucosylated variants were generated by adding 0.15 mM bait substrate 2-deoxy-2-fluoro-2-fucose to the culture medium (Dekkers et al., (2016) Sci Rep 6:36964). As shown in Example 11 below, this resulted in a significant reduction in fucose incorporation in IgG-Fc glycans.

[0229] Table 2: Antibody variants of generated anti-TNFα antibodies (EU numbering)

[0230] name Mutations relative to the parental antibody Ab-wt* None (i.e. parental antibody) Ab-REW** Q311R / M428E / N434W Ab-REW-2FF** Fucose-free variant of Ab-REW

[0231] *Antibodies not according to the invention; **Antibodies according to the invention

[0232] Antibodies Ab-REW and Ab-REW-2FF are antibodies according to the invention.

[0233] Example 1 Affinity for TNFα

[0234] method:

[0235] Affinity for TNFα was measured by Biacore. The CM5 chip was prepared using the standard amine fixation Biacore procedure. After the CM5 chip was inserted, the system was primed and then normalized using the BIA normalization solution (Biacore Preventative Maintenance Kit 2). The chip was added to the system using PBS-T running buffer; before fixation, the chip surface was primed with three injections of 50mM NaOH. Protein A was immobilized on the chip surface. To this end, the protein was diluted to 5μg / mL in 10mM acetate buffer at pH 4.5 and then injected to produce a binding reaction of approximately 1000RU in all 4 flow cells. In order to remove non-covalently bound material from all chip flow cells, three 15-second 50mM NaOH washes were performed. On the protein A chip, the antibody was captured in flow cells 2 and 4, and flow cells 1 and 3 were used for reference subtraction. The test antibody was diluted to 10nM in PBS-T and 2.5-7.5uL was injected to obtain 120RU of capture antibody. The analyte TNFα was prepared at a concentration of 500 μg / mL in water as per the supplier's instructions and further diluted into running buffer PBS-T. Single cycle kinetics were used to estimate steady-state affinity. For each single cycle analysis cycle, titrations of 5 analyte concentrations were injected over the ligand and the degree of dissociation of the complex was measured. The surface was regenerated using glycine pH 1.7. A dual reference approach was employed, in which data from the ligand-bound capture surfaces (fc 2 and 4) were subtracted from reference surfaces without captured ligand (fc 1 and 3, respectively). Blank buffer injections were run every 3-4 cycles and then subtracted from the analyte injection cycles to correct for minor variations in the ligand capture surface. Analyte injections were repeated at the beginning and end of each analytical run to check for sample degradation or changes in instrument performance. All analyses were performed at 25°C and the sample holder was incubated at 10°C during the experimental run. Each experiment was run at least 3 times. A one-to-one binding model was used to fit the resulting kinetic data.

[0236] result:

[0237] All antibodies showed similar binding kinetics to TNFα, indicating that any of the introduced modifications did not result in significant changes in the antigen binding region. For antibody Ab-REW-2FF, the off-rate could not be determined, and therefore the affinity (K D ). However, the association rate was comparable to that of other antibodies, indicating that the introduction of the mutation did not significantly affect binding.

[0238] Table 3: Binding kinetics of human IgG1 variants to TNFα determined by SPR

[0239]

[0240] * The dissociation rate (kd) could not be determined

[0241] Example 2 Efficacy

[0242] method:

[0243] 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 incubation for 20 hours at 37°C / 5% CO2, the proliferation response was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling agent (phenazine ethanol sulfate, PES). MTS is converted to formazan by dehydrogenases present in metabolically active cells. The product was measured by absorbance at 492 nm. The amount of product is proportional to the number of viable cells in culture.

[0244] result:

[0245] The results are as follows Figure 1 Introducing mutations into the Fc region of anti-TNFα antibodies did not affect potency.

[0246] Example 3 Affinity for Fcγ receptors (CD64, CD32a, CD32b, CD16a, CD16b)

[0247] method:

[0248] Affinity for FcγR was measured by Biacore. The CM5 chip was prepared using the standard amine fixation Biacore procedure. After the CM5 chip was inserted, the system was primed and then standardized using the BIA standardization solution (Biacore Preventative Maintenance Kit 2). The chip was added to the system together with phosphate buffered saline Tween-20 (PBS-T) running buffer; before fixation, the chip surface was focused on with three injections of 50mM NaOH. FcγR was immobilized on the chip surface using the His tag capture system. Anti-His tag chips were prepared according to the Biacore kit instructions, and approximately 12,000RU of antibodies were deposited in all four flow cells. In order to remove non-covalently bound material from all chip flow cells, three 30-second washes of 10mM glycine (pH 1.5) were performed. The Fcγ receptor was diluted in PBS-T to a range of 0.5-2μg / mL, and 2.5-5.0μL was injected onto the chip, resulting in capture levels between 60 and 200RU. Prior to analysis, antibodies were diluted into PBS-T. Single cycle kinetics were used to estimate steady-state affinity. For each single cycle analysis cycle, a titration of 5 antibody concentrations was injected onto the FcγR ligand and the dissociation of the complex was measured. The surface was regenerated using the recommended solution (10 mM glycine pH 1.5 for anti-His capture surface). A double reference approach was employed, in which data from ligand-bound capture surfaces (Fc 2 and 4) were subtracted from reference surfaces without capture ligand (Fc 1 and 3, respectively). For each antibody titration cycle, a blank buffer injection was performed and then subtracted from the analyte injection cycle to correct for minor variations in the ligand capture surface. All analyses were performed at 25°C and the sample holder was incubated at 10°C during the experimental run. Each experiment was run at least 3 times.

[0249] result:

[0250] The engineered anti-TNFα antibody did not affect binding to CD64. The introduction of mutations did not affect affinity for CD32a(H), CD32a(R) and CD32b. However, Ab-REW-2FF showed a 5.5-fold increase in affinity for CD16a(V). The non-fucosylated antibody Ab-REW-2FF also had improved binding to the low affinity CD16a receptor and CD16b.

[0251] Table 4: Affinity for the Fcγ receptors CD64, CD32a(H), CD32a(R) and CD32b as determined by SPR. Mean and standard deviation affinities calculated from two or more independent experiments are shown.

[0252]

[0253] Table 5: Affinity for the Fcγ receptors CD16a (V), CD16a (F) and CD16b determined by SPR. Mean and standard deviation affinities calculated from two or more independent experiments are shown.

[0254]

[0255] Example 4 Affinity for FcRn

[0256] method:

[0257] Using Biacore 3000 instrument, with CM5 sensor chip and in conjunction with anti-TNFα IgG1 antibody (about 500 resonance units (RU)), SPR is performed using the amine coupling chemistry described by the manufacturer. 2.0ug / mL of each protein is injected into 10mM sodium acetate (pH 4.5) for coupling using an amine coupling kit (GE Healthcare). pH 7.4 HBS-P buffer (10mM HEPES, 150mM NaCl, 0.005% surfactant P20) or pH 6.0 phosphate buffer (67nM phosphate buffer, 150mM NaCl, 0.005% Tween 20) is used as running and dilution buffer. Binding kinetics are determined by injecting titration (1000-31.2nM) of His-tagged monomeric human FcRn (hFcRn) on fixed antibodies at pH 7.4 or pH 6.0. All SPR experiments were performed at 25°C and a flow rate of 40 ul / min. Binding data were zeroed and reference cell values ​​were subtracted. The Langmuir 1:1 ligand binding model provided by BIAevaluation software (version 4.1) was used to determine binding kinetics.

[0258] result:

[0259] The results show that the wild-type antibody Ab-wt strictly binds to hFcRn in a pH-dependent manner. All engineered antibody variants have a higher affinity for FcRn at pH 6.0, but maintain their pH dependence and do not bind to the receptor at pH 7.4. Surprisingly, the variant containing REW shows a strong binding of >160 times at acidic pH, and no binding is detected under the conditions of the test at neutral pH. Compared with infliximab containing wild-type IgG1 Fc region, the antibody variant shows improved binding to FcRn.

[0260] Table 6: Affinity of anti-TNFα antibody variants to FcRn at pH 6.0 and pH 7.4 as determined by SPR

[0261]

[0262] NA: Not available due to weak binding

[0263] Example 5 Transcytosis

[0264] method:

[0265] A Transwell filter (1.12 cm) with a collagen-coated polytetrafluoroethylene (PTFE) membrane with a pore size of 0.4 μm was placed 2 ) were incubated O / N in complete growth medium and then inoculated with 1.00 × 10 6 T84 cells. Transepithelial electrical resistance (TEER) was monitored daily using a MILLICELL-ERS-2 voltmeter. Cultures were grown for 4-5 days and then reached approximately 1000-1300 Ω×cm 2 TEER values ​​were combined. Prior to the experiment, the monolayer was starved for 1 hour in Hank's balanced salt solution (HBSS). Then, 400nM of antibody variants or IFX were added to the top Transwell chamber alone or with 4000nM of non-specific human myeloma IgG. Samples were collected from the basolateral reservoir at 0 and 4 hours after addition. The antibody concentration in the basolateral reservoir was determined by ELISA. In brief, 96-well Maxisorp plates were O / N coated with recombinant TNFα or anti-human Fc-specific antibodies from goats, all diluted to 1μg / ml in PBS. Subsequently, the plates were blocked with PBS containing 4% skim milk for 2 hours at room temperature and then washed 4 times with PBS containing 0.05% Tween 20. Samples collected during the transcytosis experiment were added to the wells and incubated for 2 hours at room temperature and then washed as described above. 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 ALP-substrate and the absorption spectrum was recorded at 405 nm.The amount of antibody variant, IFX and total IgG transported was calculated from the standard curve for each individual antibody variant.

[0266] Transcytosis of antibody variants across polarized human epithelial cells

[0267] result:

[0268] The engineered anti-TNFα antibody variants were tested for transcytosis across cell monolayers and compared to the wt antibody or IFX, another human IgG1 anti-TNFα antibody. Figure 2As shown, the wt anti-TNFα antibody was transported from the apical to the basolateral reservoir. The transport was 2.8-fold greater for Ab-REW compared to IFX (another IgG1 antibody with a wt Fc region), and this was also the case for Ab-REW-2FF.

[0269] Transcytosis of antibody variants across polarized human epithelial cells in the presence of competing IgG

[0270] result:

[0271] When the 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 from the apical side across the polarized T84 cell monolayer to the basolateral reservoir was comparable for all antibodies. However, at pH 6.0, also in the presence of excess competing human IgG of irrelevant specificity, the increased affinity for FcRn resulted in a significantly higher percentage of specific anti-TNFα transport across the cell monolayer. Figure 3 shown.

[0272] Example 6 ADCC

[0273] method:

[0274] The ADCC reporter bioassay core kit from Promega was used. Briefly, 1 × 10 5 / mL of mTNFα CHO-K1 target cells were seeded at 100 μL per well on white (clear bottom) tissue culture plates. The plates were incubated O / N at 37°C / 5% CO2. On day 2, 95 μL of assay medium was removed and 25 μL of engineered Jurkat effector cells were added at 3×10 6 / mL replacement. The plate was then incubated at 37°C / 5% CO2 for 6 hours. At the end of the incubation period, BioGlo TM Equilibrate the plate to RT for 10-20 min, then add 75 μL BioGlo TM Reagents. After incubation in the dark for 5-10 min, luminescence was measured. The data were fitted using a 4-PL model.

[0275] result:

[0276] Results (see Figure 4 ) showed that all anti-TNFα antibodies can induce ADCC, but with different strengths. Compared with the wild-type antibody Ab-wt, the antibody variants showed increased ADCC. In particular, the non-fucosylated antibody variant Ab-REW-2FF had significantly improved ADCC.

[0277] Example 7 C1q binding

[0278] method:

[0279] ELISA was performed using 96-well MaxiSorp plates, where the wells were coated with human TNFα diluted to 1 μg / mL in PBS. After incubation O / N at 4°C, the plates were blocked with PBS containing 4% skim milk for 1 hour and washed four times with PBS containing 0.05% Tween-20 (PBS-T). Then, titrated amounts of anti-TNFα IgG antibodies were diluted in PBS-T, added and incubated for 1 hour at room temperature. After washing with PBS-T, human C1q (0.5 g / mL) was diluted in 0.1 M Veronal buffer (0.25 mM CaCl2 and 0.8 mM MgCl2 pH 7.2), added to the wells and incubated for 1 hour. Subsequently, the wells were washed as above, and then rabbit anti-human C1q diluted 1:5000 in PBS-T was added to the wells and incubated for 1 hour. After washing, HRP-conjugated anti-rabbit IgG from donkey 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. The absorbance was measured at 620 nm using a Sunrise spectrophotometer.

[0280] result:

[0281] Anti-TNFα antibody variants were captured on human TNFα before adding human C1q. Results (see Figure 5 ) show that the antibodies bind to C1q but with different binding strengths. Specifically, Ab-REW and Ab-REW-2FF bind with greater strength than IFX. The presence of fucose at the bi-antennary N-glycan attached to N297 has little or no effect on binding. The ranking of binding from strongest to weakest is as follows: Ab-REW>Ab-REW-2FF>IFX.

[0282] Example 8 CDC

[0283] method:

[0284] The anti-TNFα CDC assay measures the antibody-dependent cytotoxicity of rabbit complement. Target cells expressing mTNFα were seeded into microplates in the presence of anti-TNFα antibodies that enhance complement cytotoxic potential. Six independent samples (anti-TNFα antibody variants) and four samples of the reference standard (IFX) were prepared and serially diluted (1.3-fold dilution steps) at a concentration of 60 μg / mL and the dilution plates were sealed until use. Target cells containing the LUC viability reporter were prepared to 1.5×105 / mL and stored in a water bath at 37°C. Rabbit complement was diluted in DMEM high glucose to 3 times the final assay concentration. Complement was combined with target cells in a 1:1 ratio (v / v) immediately after preparation. 40 microliters of complement / target cell preparation was transferred to each well of the assay plate. The antibodies prepared in the dilution plate were transferred to the cells (20 μL / well) and the plates were then incubated at 36°C / 1% CO2 for 3.5 hours. The assay plate was equilibrated to RT for 35 minutes in the dark. Steady-state Glo was equilibrated to ambient temperature for 120 minutes before use, then added to the assay plate (20 μL / well) and stored in the dark at room temperature for 35 minutes before measuring luminescence. The percentage of cell death was then calculated for each concentration of each sample, and the data was fit using a 4-PL model.

[0285] result:

[0286] The results are as follows Figure 6 and Table 7. Relative EC of test sample performance in CDC analysis 50 The results for relative maximum percent death provide a clear hierarchy of activity that is consistent between both activity measurements. Ab-REW has higher CDC activity than IFX regardless of fucose content. In a direct comparison between fucose variant samples, to better understand the effect of fucose content on CDC activity, Ab-REW-2FF provided similar CDC activity responses compared to Ab-REW. The comparison shows the relative EC 50 The response was 97.1%, and the relative maximum mortality response percentage was 100.4%.

[0287] Table 7: CDC activity of anti-TNFα antibody variants compared to IFX according to relative EC50 and relative percentage of death performance

[0288]

[0289] Example 9 Induction of regulatory macrophages

[0290] method:

[0291] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy buffy coats. Cells were separated by Ficoll gradient centrifugation. Equal amounts of cells from two separate donors were mixed and 2 x 10 5Cells were plated in a 96-well plate with a total volume of 100 μL per 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 a final concentration of 10 μg / mL. Each compound was added five or six times in duplicate. The final volume was 150 μL / well. Human serum IgG1 (Sigma#I5154) was used as a control. After the addition of the compound, the mixed lymphocyte reaction (MLR) was cultured for another 4 days at 37°C / 5% CO2. Afterwards, the plate was washed with PBS / 5mM EDTA (PBS / EDTA) and incubated with 50 μL / well of PBS / EDTA at RT for 20 minutes. The plate was centrifuged and the liquid was discarded. The antibodies were diluted in PBS / EDTA (anti-CD14-PE, anti-CD206-APC, both diluted 1:10). The cells were resuspended in 50 μL of antibody solution and incubated at RT for 20 minutes. Afterwards, cells were washed with PBS / EDTA and resuspended in 50 μL PBS / EDTA. Stained samples were analyzed on a FACS Fortessa using FACSDiva software. Analysis was performed using FlowJo software.

[0292] result:

[0293] The induction of regulatory macrophages was analyzed in four independent MLRs and was successful in all experiments (IFX was compared to IgG control). Figure 7 As shown. Since each experiment was performed using different donors with individual differences, the level of IFX induction may vary between experiments. All tested anti-TNFα antibody variants induced CD14 + CD206 + Regulatory macrophages, with slight variation between compounds. Ab-REW and Ab-REW-2FF induced slightly more regulatory macrophages than IFX, however, only in the case of Ab-REW-2FF was this increase significant.

[0294] Example 10 Inhibition of T cell proliferation

[0295] method:

[0296] PBMCs were isolated from healthy buffy coats. Cells were separated by Ficoll gradient centrifugation. Equal numbers of cells from two separate donors were mixed and 2 x 10 5Cells were seeded in a 96-well plate in a total volume of 100 μL per 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 a final concentration of 10 μg / mL. Each compound was added in quintuples. The final volume was 150 μL / well. Human serum IgG1 (Sigma#I5154) was used as a control. After the addition of the compound, the mixed lymphocyte reaction (MLR) was cultured for another 2 days at 37°C / 5% CO2. Then, tritiated thymidine (3H thymidine, 0.5 microcuries / well) was added to the culture. The culture was further incubated at 37°C / 5% CO2 for 18 hours. Samples were collected using a Microbeta Filtermat 96 cell collector and analyzed using a Microbeta MicroplateCounter equipped with a single detector. The samples were counted for 10 seconds / well and converted to counts per minute (cpm).

[0297] result:

[0298] Inhibition of T cell proliferation was measured in three independent MLRs and success was defined as inducing inhibition with IFX as a positive control. The level of inhibition with IFX is likely to vary between individual experiments due to differences in the induction of regulatory macrophages. In each experiment, the potential of the anti-TNFα antibody variants to inhibit T cell proliferation was calculated relative to the positive control IFX. Antibody Ab-REW-2FF showed significantly enhanced inhibition compared to IFX, whereas Ab-REW showed comparable inhibition to IFX (see Figure 2). Figure 8 ).

[0299] Example 11 N-glycan analysis

[0300] method:

[0301] 50 μl of each IgG variant (1 mg / ml) was centrifuged at 13,000 x g for 10 minutes and incubated at 37 ° C overnight before adding 1 μg trypsin dissolved in 100 μl 50mM ammonium bicarbonate (pH 7.8). The centrifugal device was centrifuged at a speed of 13,000 x g for 10 minutes, and the flow-through was transferred to an Eppendorf tube and dried in a SpeedVac (Heto Maxi drying). The dried sample was dissolved in 20 μl 1% formic acid, sonicated for 30 s, and then centrifuged at 16,100 x g for 10 minutes. Subsequently, each sample was transferred to a new sample bottle and the proteolytic peptides were analyzed by reverse phase (C18) nano online liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a Dionex Ultimate 3000UHPLC system (ThermoFisher Scientific, USA). 5 μl of peptide solution was injected into the extraction column, and the peptides were eluted from the extraction column onto the analytical column in back-flush 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 of 3% to 50% acetonitrile in water for 60 min at a flow rate of 0.3 μl / min. The LC system was coupled to a Q Precision Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) via a nano-electrospray ion source. The peptide samples were analyzed using a high energy collision dissociation (HCD) fragmentation method with a normalized collision energy of 20, and an Orbitrap survey scan was obtained in the mass range of m / z 300-2000, followed by MS / MS of the ten most intense ions in the Orbitrap.

[0302] Data analysis was performed on Xcalibur v2.0. MS / MS spectra of all N-glycopeptides were extracted by oxygen ion search; 204.086 (N-acetylhexosamine) and 366.1388 (N-acetylhexose-hexose). Glycan structure and peptide mass of IgG were detected by using HCD fragment with normalized collision energy at 20. Extracted ion chromatograms of target diol peptides (EEQYNSTYR for IgG1) were extracted with an accuracy of 10 ppm and the corresponding MS / MS spectra were manually verified. Peptide sequences were detected using HCD fragment with normalized collision energy at 35 and peptide mass was verified to correspond to the correct peptide sequence. The area under the curve was calculated for all extracted diol peptides and the percentage of each glycoform was determined.

[0303] result:

[0304] For Ab-REW, two N-glycan forms predominated and corresponded to >90% of the total N-glycan pool, namely 4GlcNac-1Fuc-3Man and 4GlcNac-1Fuc-3Man-1Gal. Both dominant N-glycan forms contained core fucose. To generate a “non-fucosylated” form of Ab-REW (Ab-REW-2FF), the bait substrate 2-deoxy-2-fluoro-1-fucose (2FF) was used. MS mapping of this antibody showed that this strategy successfully led to a significant reduction in the incorporation of fucose, as a drop from >90% to 13% was detected. The major N-glycan forms after treatment were identical to the variants generated in the absence of 2FF, except that these structures lacked fucose (see also Fig.10 ).

[0305] Table 8: Percentage of N-glycan forms attached to N297 of anti-TNFα IgG antibodies

[0306]

Claims

1. An antibody comprising a TNFα binding domain and an FcRn binding site, having a high affinity for human FcRn at pH 6, wherein the high affinity is characterized by a dissociation equilibrium constant (K D ) is less than 100 nM, and further has no affinity or low affinity for human FcRn at pH 7.4, wherein the low affinity is characterized by a K D greater than 10 μM, wherein amino acids 311R, 428E and 434W according to the EU numbering system are introduced into the Fc region of the antibody, and wherein the antibody comprises (i) V L A domain comprising a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 14, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 15, and a CDR3 region consisting of the amino acid sequence shown in SEQ ID NO: 16, and (ii) V H A domain comprising a CDR1 region consisting of the amino acid sequence shown in SEQ ID NO: 17, a CDR2 region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a CDR3 region consisting of the amino acid sequence shown in SEQ ID NO:

19.

2. The antibody according to claim 1, which has an affinity for human FcRn at pH 6 that is greater than the affinity of infliximab for human FcRn at pH 6.

3. The antibody of claim 1 or 2, wherein the high affinity for human FcRn at pH 6 is characterized by a dissociation constant K D Less than 10nM.

4. The antibody according to claim 1, which has a K of less than 100 pM. D Binds human TNFα.

5. The antibody of claim 1, which is transported across a polarized cell monolayer from the apical side to the basolateral side in greater amounts than infliximab.

6. The antibody of claim 5, 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.

7. The antibody of claim 1, wherein in the presence of a ten-fold excess of competing immunoglobulin, a greater percentage of the antibody is transported across a polarized cell monolayer from the apical side to the basolateral side than infliximab, wherein the percentage refers to the total mass of immunoglobulin transported across the polarized cell monolayer.

8. The antibody of claim 7, wherein the percentage of antibody transported across the polarized cell monolayer is greater than three times the percentage of infliximab transported across the polarized cell monolayer. 9 . The antibody according to claim 1 , which is a non-fucosylated antibody or an antibody with reduced fucosylation.

10. Use of the antibody according to claim 1 in preparing a medicament for treating inflammation.

11. The use according to claim 10, wherein The inflammation is gastrointestinal inflammation.

12. The use according to claim 10, wherein The treatment comprises oral administration of an effective amount of the antibody.

13. The use according to claim 10, wherein The antibody is administered topically.

14. A pharmaceutical composition comprising the antibody according to claim 1.

15. An in vitro method for improving the transcytosis of the antibody as defined in claim 1, comprising introducing the substitutions Q311R, M428E and N434W in the amino acid sequence of the Fc region of the antibody.

16. An in vitro method for extending the plasma half-life of the antibody as defined in claim 1, comprising introducing the substitutions Q311R, M428E and N434W in the amino acid sequence of the Fc region of the antibody.

17. The antibody according to claim 1, comprising a V having an amino acid sequence as shown in SEQ ID NO:

20. H A region and a V region having an amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22 L district. 18 . The antibody according to claim 1 , 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.

19. The antibody according to claim 1, wherein the antibody specifically binds to human TNFα.

Images