Antibody variants

Abstract

The present invention relates to an antibody that binds to TNF [alpha] and exhibits modified FcRn binding. The antibody of the invention has good effector function and / or pharmacokinetic characteristics.

Description

[0001] This application is a divisional application of the Chinese invention patent application (application date: September 11, 2018; application number: 201880060827.8 (International application number: PCT / EP2018 / 074523); invention name: Antibody variant). Technical Field

[0002] The present invention relates to modified antibodies with improved effector function and / or pharmacokinetic properties. The antibodies can be used for therapeutic treatment of various disorders, particularly inflammatory conditions. Background Art

[0003] In 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, thereby producing humanized or even fully human antibodies. Another approach is to optimize antibodies by improving their effector functions. Direct effects are mediated by the variable antigen binding regions of antibodies, while indirect effects are mediated by constant Fc regions. Efforts to improve effector functions are mainly focused on regulating the Fc region. In addition, it is desirable to improve the serum half-life of therapeutic antibodies, which can reduce the amount of required antibodies and can improve their convenience for patients by extending the treatment interval.

[0004] For therapeutic applications, immunoglobulin G (IgG) has been the preferred class of choice for several reasons; IgG is easy to purify, relatively stable in storage, can be administered intravenously, has a prolonged biological half-life in vivo, and is able to participate in a range of biological effector functions, such as activation of complement-dependent cytotoxicity (CDC) and recruitment of effector cells through various Fc receptor interactions (antibody-dependent cellular cytotoxicity; ADCC). Of the five immunoglobulin classes, IgG exhibits the longest biological half-life due to its unique interaction with the IgG recycling receptor, the neonatal Fc receptor (FcRn). One of the known functions of the receptor is to rescue IgG from catalytic degradation. The solved FcRn-Fc co-crystal structure has shown that the interaction with Fc occurs at the IgG hinge-C H 2-C H -3 zone. This interaction occurs in the endosome 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, where they are released into the circulation at a physiological pH of 7.4, while the non-complexed IgG molecules are destined for lysosomal degradation. This recycling is the mechanism for achieving an extended half-life of IgG; regulation of the FcRn-IgG interaction will therefore 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 retention time of IgG. For therapeutic applications, a longer half-life is desirable because less dosage and less injection will be required. Several methods for increasing the half-life have been studied, including the use of polyethylene glycol (PEG), the generation of albumin or Fc fusion proteins, and the enhancement of FcRn-IgG interactions. Pegylated agents have been in clinical use since 1990, and Pegylation is an established technology for extending the drug retention in the blood. Because human serum albumin (HSA) is also recycled by FcRn via pH-dependent interactions, several albumin-fusion proteins that enhance stability and half-life have also been prepared. In addition, antibody fragments fused to albumin or albumin binding domains have been shown to extend serum retention time in preclinical studies. Producing Fc fusion proteins is another strategy that will confer similar properties to proteins or peptides and intact antibodies.

[0006] Fc region modifications that have been investigated are summarized in Saxena (2016) Frontiers in Immunology, Volume 7, Article Number 580. Various Fc mutations are further described in WO 1998 / 023289 A1, WO 2000 / 042072 A2, WO 2010 / 106180 A2 and WO 2014 / 108198 A1.

[0007] There is 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 an antibody increase the affinity of the antibody to FcRn at pH 6, while the affinity at pH 7.4 remains low. Antibodies with these mutations have improved pharmacokinetic properties. In addition, the preferred antibody variants of the present invention have better inhibition of T cell proliferation than their corresponding parent wild-type antibodies.

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

[91] :

[0010] [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 500 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 1 μM, wherein the amino acid sequence of the antibody comprises amino acids 380A and 434A.

[0011] [2] The antibody according to item [1], wherein the antibody is obtained by substituting asparagine at position 434 with alanine (N434A).

[0012] [3] The antibody according to item [1] or [2], wherein the antibody is obtained by substituting glutamic acid at position 380 with alanine (E380A).

[0013] [4] The antibody according to any one of the preceding items, wherein the amino acid at position 307 of the amino acid sequence of the antibody is other than alanine.

[0014] [5] The antibody according to any one of the preceding items, wherein the amino acid sequence of the antibody further comprises amino acid 307T.

[0015] [6] The antibody according to any one of the preceding items, comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 13.

[0016] [7] The antibody according to any one of the preceding items, wherein the affinity of the antibody for human FcRn at pH 6 is greater than that of infliximab (IFX).

[0017] [8] 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 400nM.

[0018] [9] 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 300nM.

[0019]

[10] 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 200nM.

[0020]

[11] 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 150nM.

[0021]

[12] 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 In the range of 5nM to 500nM, or 10nM to 400nM, or 25nM to 300nM, or 50nM to 200nM, or 75nM to 175nM.

[0022]

[13] The antibody according to any one of the preceding items, wherein the K value characterizing the high affinity at pH 6 is D It was measured by surface plasmon resonance (SPR).

[0023]

[14] The antibody according to any one 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 one of the preceding items, wherein the K value characterizing the low affinity for human FcRn at pH 7.4 is D It was measured by SPR.

[0025]

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

[13] , wherein the affinity for human FcRn at pH 7.4 is too low such that K D Values ​​could not be determined by SPR.

[0026]

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

[0027]

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

[0028]

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

[0029]

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

[0030]

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

[0031]

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

[0032]

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

[0033]

[24] 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 an amount greater than that of infliximab.

[0034]

[25] The antibody of item

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

[0035]

[26] The antibody of any one of items

[23] to

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

[0036]

[27] An antibody as described in any of items

[22] to

[26] , wherein the amount of the antibody transported across the polarized cell monolayer is twice as great as 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 as described in any of items

[22] to

[27] , 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 it contains amino acids 380E and 434N.

[0038]

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

[0039]

[30] The antibody of item

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

[0040]

[31] An antibody as described in item

[29] or

[30] , wherein the percentage of the antibody transported across polarized cell monolayers is two times greater than the percentage of the parent immunoglobulin transported across polarized cell monolayers, wherein the parent immunoglobulin differs from the antibody only in that it contains amino acids 380E and 434N.

[0041]

[32] The antibody of any one of items

[22] to

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

[0042]

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

[0043]

[34] An antibody as described in any of the above items, which has a K of less than 10 μM D Binds to CD32a(H).

[0044]

[35] An antibody as described in any of the above items, which has a K of less than 10 μM D Binds to CD32a(R).

[0045]

[36] An antibody as described in any of the above items, which has a K of less than 10 μM D Binds to CD32b.

[0046]

[37] An antibody as described in any of the preceding items, which has a K of less than 1000 nM, preferably less than 100 nM D Binds to CD16a(V).

[0047]

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

[0048]

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

[0049]

[40] The antibody according to any one of the preceding items, which is capable of binding to human C1q.

[0050]

[41] The antibody according to any one of the preceding items, which has complement-dependent cytotoxicity (CDC) of rabbit complement.

[0051]

[42] The antibody according to any one of the preceding items, which has antibody-dependent cellular cytotoxicity (ADCC).

[0052]

[43] The antibody according to any one of the preceding items, which is capable of inducing CD14+CD206+ macrophages.

[0053]

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

[0054]

[45] The antibody according to any one of the preceding items, which is capable of inhibiting T cell proliferation.

[0055]

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

[0056]

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

[0057]

[48] ​​The antibody according to any one of the preceding items, comprising: (i) V L domain, the V L The structural domain comprises 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, and a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 5; and (ii) V H domain, the V H The domain comprises 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.

[0058]

[49] The antibody according to any one of the preceding items, comprising a V having an amino acid sequence as shown in SEQ ID NO: 9 H domain and a V domain having an amino acid sequence as shown in SEQ ID NO: 10 L Structural domain.

[0059]

[50] The antibody according to any one of the preceding items, comprising a light chain having the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 11.

[0060]

[51] The antibody of any one of items [1] to

[47] , wherein the antibody comprises: (i) V L domain, the V L The domain comprises 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, and a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 16; and (ii) V H domain, the V HThe domain comprises 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.

[0061]

[52] The antibody according to item

[51] , comprising a V having an amino acid sequence as shown in SEQ ID NO: 20. H domain and a V domain having an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22 L Structural domain.

[0062]

[53] The antibody as described in item

[51] or

[52] , 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.

[0063]

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

[0064]

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

[0065]

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

[0066] (i) with a dissociation constant (K) of less than 125 pM D ) binds to human TNFα;

[0067] (ii) cross-reacts with rhesus TNFα and cynomolgus TNFα;

[0068] (iii) having a potency greater than that of infliximab as determined by the L929 assay; and / or

[0069] (iv) capable of being expressed in a stoichiometry of at least 2 (antibody: TNFα 三聚体 ) and human TNFα 三聚体 Combine.

[0070]

[57] An antibody as described in any of the above items, which has a K of less than 1 nM D Binds to TNFα from rhesus monkey.

[0071]

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

[0072]

[59] An antibody as described in any of the preceding items, wherein the potency of the antibody in inhibiting TNFα-induced apoptosis relative to the potency of infliximab (relative potency) is greater than 3 as measured in the L929 assay, and wherein the relative potency is the IC of infliximab in ng / mL in the L929 assay. 50 Values ​​are compared to the IC values ​​of the antibodies in ng / mL in the L929 assay. 50 The ratio of values.

[0073]

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

[0074]

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

[0075]

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

[0076]

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

[0077]

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

[0078]

[65] The antibody according to any one of the above items, which is capable of inhibiting LPS-induced CD14 + Interleukin-1β secretion by monocytes.

[0079]

[66] The antibody according to item

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

[0080]

[67] The antibody according to item

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

[0081]

[68] The antibody according to any one of the preceding items, which is capable of inhibiting LPS-induced CD14 + TNFα secretion by monocytes.

[0082]

[69] The antibody according to item

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

[0083]

[70] The antibody according to item

[69] , wherein the IC of the inhibition of LPS-induced TNFα secretion is 50 The value is less than the IC of adalimumab 50 value.

[0084]

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

[0085]

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

[0086]

[73] A vector or plasmid comprising the nucleic acid according to item 72.

[0087]

[74] A cell comprising the nucleic acid of item

[72] or the vector or plasmid of item

[73] .

[0088]

[75] A method for preparing the antibody described in any one of items [1] to

[71] , comprising culturing the cell described in item

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

[0089]

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

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

[0090]

[77] The antibody for use according to item

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

[0091]

[78] The antibody for use according to item

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

[0092]

[79] The antibody for use according to item

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

[0093]

[80] The antibody for use according to item

[78] or

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

[0094]

[81] The antibody for use according to item

[80] , wherein the Crohn's disease is selected from the group consisting of ileum, colon, ileocolonic, and / or isolated upper Crohn's disease (gastric, duodenal and / or jejunal) and includes non-stricturing / non-penetrating, stricture, penetrating and perianal disease behaviors, thereby allowing any combination of localization and disease behavior of any of the above mentioned.

[0095]

[82] The antibody for use according to item

[78] or

[79] , wherein the inflammatory disorder of the gastrointestinal tract is ulcerative colitis.

[0096]

[83] The antibody for use according to item

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

[0097]

[84] The antibody for use according to item

[78] or

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

[0098]

[85] The antibody for use according to item

[76] , wherein the inflammatory disorder is arthritis.

[0099]

[86] The antibody for use according to item

[76] or

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

[0100]

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

[76] to

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

[0101]

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

[76] to

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

[0102]

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

[71] .

[0103]

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

[0104]

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

[0105] The present disclosure also relates to the following embodiments:

[0106] 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 500 nM, and it further has no affinity or low affinity for human FcRn at pH 7.4, the low affinity being characterized by a K D Greater than 10 μM, wherein the amino acid sequence of the antibody comprises amino acids 380A and 434A.

[0107] 2. The antibody according to embodiment 1, wherein the amino acid sequence of the antibody further comprises amino acid 307T.

[0108] 3. The antibody according to any one of the preceding embodiments, whose affinity for human FcRn at pH 6 is greater than the affinity of infliximab for human FcRn at pH 6.

[0109] 4. The antibody according to any one of the preceding embodiments, wherein the high affinity for human FcRn is characterized by a dissociation equilibrium constant (K D ) is less than 300nM.

[0110] 5. The antibody according to any one of the preceding embodiments, wherein the antibody has a K of less than 100 pM. D Binds to human TNFα.

[0111] 6. The antibody according to any of the preceding embodiments, which is transported across polarized cell monolayers from the apical side to the basolateral side in an amount greater than infliximab.

[0112] 7. An antibody according to embodiment 6, wherein the amount of the antibody transported across the polarized cell monolayer is twice as great as the amount of the parent immunoglobulin transported across the polarized cell monolayer, wherein the parent immunoglobulin differs from the antibody only in that it contains amino acids 380E and 434N.

[0113] 8. An antibody according to any of the preceding embodiments, wherein in the presence of a ten-fold excess of competing immunoglobulin, a greater percentage of the antibody than the percentage of infliximab is transported across a polarized cell monolayer from the apical side to the basolateral side, wherein the percentage refers to the total mass of the immunoglobulin transported across the polarized cell monolayer.

[0114] 9. The antibody according to any one of the preceding embodiments, which is a non-fucosylated antibody.

[0115] 10. A nucleic acid encoding the antibody according to any one of the preceding embodiments.

[0116] 11. The antibody according to any one of embodiments 1 to 9, for use in treating an inflammatory condition.

[0117] 12. The antibody for use according to embodiment 11, wherein the inflammatory condition is an inflammatory disorder of the gastrointestinal tract.

[0118] 13. The antibody for use according to embodiment 11, wherein the treatment comprises oral administration of an effective amount of the antibody.

[0119] 14. The antibody for use according to embodiment 11 or 12, wherein the antibody is administered topically.

[0120] 15. A pharmaceutical composition comprising the antibody according to any one of embodiments 1 to 9.

[0121] 16. A method for improving the endocytic transport of an antibody against TNFα, the method comprising introducing substitutions E380A and N434A into the amino acid sequence of the antibody.

[0122] 17. A method for prolonging the plasma half-life of an antibody against TNFα, the method comprising introducing substitutions E380A and N434A into the amino acid sequence of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 : Potency of anti-TNFα antibody variants in neutralizing human TNFα in the L929 assay. Dose response curves for TNFα-specific TP antibodies and the reference infliximab are shown.

[0124] Figure 2 : Transport of anti-TNFα IgG variants across polarized T84 cells. Amounts of anti-TNFα antibody variants and IFX from apical to basolateral reservoirs 4 hours after addition. Expressed as ng / cm 2 Error bars indicate SD of two to four individual monolayers.

[0125] Figure 3 : Transport of anti-TNFα IgG variants across polarized T84 cells in the presence of excess myeloma IgG. Amounts of anti-TNFα IFX and Ab variants transported from the apical to the basolateral reservoir in the presence of a 10-fold excess of human myeloma IgG 4 hours after addition. Expressed as ng / cm 2 Error bars indicate SD of three to four individual monolayers.

[0126] Figure 4 : ADCC activity. Induction of ADCC by anti-TNFα antibody variants and wild-type antibodies.

[0127] 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 indicate SD.

[0128] Figure 6 : The effect of each compound on CD14 relative to the induction of IFX + CD206 + Induction of macrophages. Pooled data from 4 independent experiments. Bars represent mean values ​​and error bars represent SEM.

[0129] Figure 7 : Inhibition of T cell proliferation by each compound relative to IFX. Pooled data from 3 independent experiments. Bars represent mean values ​​and error bars represent SEM.

[0130] Figure 8 : Schematic diagram of site-directed mutagenesis.

[0131] Fig. 9 : Schematic representation of the dominant N-glycan forms attached to N297 of anti-TNFα antibody variants. The two N-glycan profiles that dominated the panel of anti-TNFα antibody variants tested were 4GlcNac-1Fuc-3Man and 4GlcNac-1Fuc-3Man-1Gal, while for IgG variants prepared in the presence of 2FF, the same biantennary structures appeared, but these lacked fucose. DETAILED DESCRIPTION

[0132] The present invention relates to an antibody that can bind to TNFα and comprises an FcRn binding site. According to the present application, if the antibody can bind to FcRn, preferably to human FcRn, at pH 6, it comprises an FcRn binding site. Binding to FcRn at pH 6 can be determined by SPR, 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, and the high affinity is characterized by a dissociation equilibrium constant (K D ) is less than 500 nM. The antibodies of the present invention further have a low affinity for human FcRn at pH 7.4, characterized by a K D Less than 1 μM. The amino acid sequence of the antibody comprises amino acids alanine at position 380 and position 434 (EU numbering).

[0133] Throughout the specification and claims, 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 variable domain (roughly, residues 1-107 of the light chain and residues 1-113 of the heavy chain). The "EU numbering system" or "EU index" is generally used when referring to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference). Unless otherwise stated herein, reference to residue numbers in the variable domain of an antibody means the residue number obtained by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in an antibody constant domain refer to the residue numbers obtained by the EU numbering system (see, eg, WO 2006 / 073941).

[0134] Antibody

[0135] 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 maintains one or more properties of the parent antibody. 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.

[0136] 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 organism, prokaryotic organism or phage clone, rather than a method for producing the antibody. Monoclonal antibodies can be prepared using a variety of techniques known in the art (including the use of hybridoma, recombinant and phage display technology or a combination thereof). (Harlow and Lane, "Antibodies, A Laboratory Manual" CSH Press 1988, Cold Spring Harbor NY).

[0137] In other embodiments (including embodiments involving in vivo use of anti-TNFα antibodies in humans), chimeric, primatized, 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.

[0138] In another specific embodiment, the antibody of the present invention is an immunoglobulin, preferably immunoglobulin G (IgG). The subclasses of the IgG of the present invention are not limited to and include IgG1, IgG2, IgG3, and IgG4. Preferably, the IgG of the present invention belongs to subclass 1, 2 or 4, i.e., it is an IgG1, IgG2, or IgG4 molecule, respectively. Most preferably, the IgG of the present invention belongs to subclass 1, i.e., it is an IgG1 molecule.

[0139] TNFα binding domain

[0140] 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.

[0141] Preferably, the antibodies of the present invention specifically bind to TNFα. As used herein, an antibody "specifically recognizes" human TNFα, 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 50The term "specific binding" refers to the ability of an antibody to distinguish between human TNFα and unrelated biomolecules, for example, as determined by specific assay methods known in the art. Such methods include, but are not limited to, immunoblotting and ELISA tests. For example, standard ELISA assays can be performed. Typically, the determination of binding specificity is performed by using not a single reference biomolecule, but a group of about three to four unrelated molecules (such as milk powder, BSA, transferrin, etc.). In one embodiment, specific binding refers to the ability of an antibody to distinguish between human TNFα and human TNFβ.

[0142] 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.

[0143] The term "CDR" refers to one of the six hypervariable regions that mainly contribute to antigen binding in the variable domain of an antibody. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication No. 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. However, the CDR1 (CDR H1 or H1) of the heavy chain variable domain as used herein is defined by the following residues (Kabat numbering): it starts at position 26 and ends before position 36.

[0144] In one embodiment of the invention, the antibody of the invention is an anti-TNFα antibody as disclosed in any one of the originally filed PCT applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 and PCT / EP2017 / 056227. In yet another embodiment of the invention, the antibody is an anti-TNFα antibody having a light chain variable domain and / or a heavy chain variable domain comprising complementary determining regions (CDRs) having amino acid sequences as disclosed in the originally filed PCT applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 and PCT / EP2017 / 056227.

[0145] In a preferred embodiment of the invention, the antibody is an anti-TNFα antibody having a light chain variable domain and / or a heavy chain variable domain comprising one or more CDRs, the complementarity determining regions having amino acid sequences as disclosed in PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237, or PCT / EP2017 / 056227. In another preferred embodiment of the invention, the antibody is an anti-TNFα antibody having a light chain variable domain and a heavy chain variable domain comprising CDRs having amino acid sequences 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 yet another preferred embodiment of the present invention, the anti-TNFα antibody is selected from the group consisting of: an anti-TNFα antibody comprising the heavy chain variable domain amino acid sequence and / or the light chain variable domain amino acid sequence according to claim 4 of PCT / EP2017 / 056218, claims 5 and 6 of PCT / EP2017 / 056246, claims 5 and 6 of PCT / EP2017 / 056237, claim 4 of PCT / EP2017 / 056227, and combinations thereof. The 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 form part of the disclosure of the present application.

[0146] In a specific embodiment, the antibody of the present invention comprises: (i) V L domain, the VL The structural domain comprises 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, and a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 5; and (ii) V H domain, the V H The domain comprises 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.

[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: 9. H In another more preferred embodiment, the antibody comprises a V domain 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 domain having an amino acid sequence as shown in SEQ ID NO: 9; H domain and (ii) a V domain having an amino acid sequence as shown in SEQ ID NO: 10 L Structural domain.

[0148] In another specific embodiment, the antibody of the present invention comprises: (i) V L domain, the V L The domain comprises 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, and a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 16; and (ii) V H domain, the V H The domain comprises 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.

[0149] 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 domain having an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22. LMost preferably, the antibody of the present invention comprises: (i) a V domain having an amino acid sequence as shown in SEQ ID NO: 20; H domain and (ii) a V domain having an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22 L Structural domain.

[0150] The antibodies of the present invention have a high affinity for human TNFα. D ” refers to the dissociation equilibrium constant of the antibody-antigen interaction. Generally, the antibodies of the invention dissociate at a rate of less than about 2×10 -10 M, preferably less than 1.5x10 -10 M, preferably less than 1.25x10 -10 M, preferably less than 1x10 -10 M, preferably less than 7.5x10 -11 M or even smaller than 5x10 -11 The dissociation equilibrium constant (K D ) binds to human TNFα as determined using surface plasmon resonance (SPR) technology in a BIACORE instrument. Specifically, K was performed as described in Example 1 D Determination of.

[0151] Modifications that affect affinity for FcRn

[0152] The antibodies of the present invention comprise an amino acid sequence that is different from the amino acid sequence of the native sequence of the wild-type antibody by means of at least one "amino acid modification" as defined in the text. The at least one amino acid modification affects the affinity of the antibody to human FcRn. Typically, at least one amino acid modification increases the affinity of the antibody to human FcRn at pH 6. In one embodiment, at least one amino acid modification increases the affinity of the antibody to human FcRn at pH 6, wherein it does not significantly change the affinity to human FcRn at pH 7.4. Preferably, the modified antibody has at least one amino acid substitution compared to the amino acid sequence of the wild-type antibody or parent antibody, such as about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions. 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 located outside the FcRn binding site of the antibody, which, for example, affect FcRn binding through structural changes. The amino acid modification(s) can be produced by methods known per se, for example by site-directed mutagenesis as described in “Antibody Engineering—Methods and Protocols”, edited by Patrick Chames, 2nd edition, 2012, Chapter 31 (ISBN 978-1-61779-973-0).

[0153] The antibodies of the present invention comprise amino acids alanine at position 380 and at position 434 (EU numbering). This is referred to herein as "380A" and "434A". The native amino acid at position 380 of an unmodified human IgG antibody is glutamic acid (E). The native amino acid at position 434 of an unmodified human IgG antibody is asparagine (N). Therefore, the antibodies of the present invention can be obtained by introducing mutations E380A and N434A into antibodies. Preferably, the antibodies of the present invention can be obtained by or by substituting glutamic acid at position 380 with alanine, and substituting asparagine at position 434 with alanine.

[0154] The remaining amino acid sequence of the Fc domain may be identical to the native amino acid sequence of a typical human IgG. However, it is possible that the amino acid sequence of the antibody comprises one or more additional mutations or substitutions to the native amino acid sequence of the Fc region of a native antibody, as long as the antibody still has TNFα binding activity, FcRn binding activity at pH 6.0, and one or more effector functions.

[0155] In a preferred embodiment, the Fc region (including the hinge region) of the antibody of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO:13.

[0156] 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.

[0157] 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.

[0158] In a preferred aspect of the invention, the antibody of the invention is a non-fucosylated antibody or an antibody with reduced fucosylation.

[0159] As used herein, the term "antibody with reduced fucosylation" refers to an antibody in which less than 90% of the N-glycans of the antibody 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 10 of the present application.

[0160] In one embodiment, less than 75%, or less than 50%, or less than 25% of the N-glycans of the antibody are fucosylated. Most preferably, less than 10% of the N-glycans of the antibody are fucosylated. In a specific embodiment, the N-glycans of the antibody of the invention do not contain any fucose.

[0161] Preferably, less than 90% of the N-glycans at N297 (EU numbering) of the antibody are fucosylated. In another embodiment, less than 75%, or less than 50%, or less than 25% of the N-glycans at N297 (EU numbering) of the antibody are fucosylated. Most preferably, less than 10% of the N-glycans at N297 (EU numbering) of the antibody are fucosylated.

[0162] In another embodiment, the antibody's N-glycan at N297 does not contain any fucose.

[0163] Non-fucosylated antibodies (sometimes also referred to as afucosylated antibodies) can be produced by various methods. For example, the coordinated knockdown of genes for α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in CHO cells can be used to prepare monoclonal antibody variants that are completely afucosylated and have enhanced ADCC (see, e.g., Imai-Nishiya et al. (2007) BMC Biotechnol. 7, 84). Using zinc finger nucleases (ZFNs), the FUT8 gene in the region encoding the catalytic core of α1,6-fucosyltransferase and thus destroying the corresponding enzyme function in CHO cells can be used to prepare monoclonal antibodies that are completely lacking in core fucose (see, e.g., Malphettes et al. (2010) Biotechnol. Bioeng. 106, 774-783).

[0164] 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), thereby reducing the incorporation of fucose into IgG-Fc glycans.

[0165] In another embodiment, the antibodies of the invention have a high sialic acid content. Increasing 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 α2,3-sialyltransferase (see, e.g., Son et al. (2011) Glycobiology 21, 1019-1028).

[0166] Affinity for FcRn

[0167] The antibodies of the present invention have a high affinity for human FcRn at pH 6. The high affinity binding of the antibody to human FcRn at pH 6 is characterized by K D Preferably, the K for high affinity binding at pH 6 is less than 500 nM. D The value is less than 400 nM, or less than 300 nM, or less than 200 nM. For example, the K value characterizing the affinity at pH 6 D Values ​​may range from 5 to 500 nM, or from 10 to 400 nM, or from 25 to 300 nM, or from 50 to 200 nM, or from 100 to 150 nM.

[0168] In a preferred embodiment, the affinity of the antibody of the present invention for human FcRn at pH 6 is greater than the affinity of infliximab for human FcRn at pH 6.0.

[0169] 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.

[0170] The antibodies of the invention generally have low affinity for human FcRn at pH 7.4. The low affinity is indicated by a K of greater than 1 μM. 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.

[0171] In a specific embodiment, the low affinity at pH 7.4 is so low that the K D Values ​​could not be determined by SPR.

[0172] In one embodiment, (i) the K of an antibody of the invention binding to human FcRn at pH 7.4 is D (ii) K for binding to human FcRn at pH 6.0 D The ratio of the values ​​is at least 50. Preferably, the ratio is at least 100, or at least 150, or at least 200.

[0173] Functional properties of antibodies

[0174] The antibodies of the present invention are effectively transported across polarized cell monolayers from the apical side 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 to infliximab refers to mass / cm 2Relative to the amount of infliximab transported across the polarized cell monolayer, the amount of antibody transported across the polarized cell monolayer is at least 110%, preferably at least 120%, more preferably at least 130%, or at least 140%, or at least 150% (wherein the amount of infliximab transported is set to 100%).

[0175] 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.

[0176] 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 is greater than the percentage of infliximab transported across the polarized cell monolayer from the apical side to the basolateral side. Relative to the percentage of infliximab transported across the polarized cell monolayer in the presence of a 10-fold excess of an irrelevant antibody, 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 120%, or at least 130%, or at least 140%, or at least 150% (infliximab is set at 100%).

[0177] Preferably, the polarized cell monolayer is a monolayer of polarized T84 cells.A transport assay mimicking the transcytotic transport process may be performed as described in Example 5 of the present application.

[0178] The antibodies of the present invention bind to CD64, CD32a(H), CD32a(R), CD32b, CD16a(V), CD16a(F) and CD16b(NA2).

[0179] The antibodies of the present invention generally have a K of less than 100 nM, preferably less than 10 nM. D Binds to CD64.

[0180] The antibodies of the present invention generally have a K of less than 10 μM. D Binds to CD32a(H).

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

[0182] The antibodies of the present invention generally have a K of less than 10 μM. D Binds to CD32b.

[0183] The antibodies of the present invention are typically, for example, less than 1 μM, preferably less than 500 nM, more preferably less than 100 nM. D Binds to CD16a(V).

[0184] The antibodies of the present invention are generally present, for example, with a K of less than 10 μM, preferably less than 1 μM. D Binds to CD16a (F).

[0185] The antibodies of the present invention are generally present, for example, with a K of less than 10 μM, preferably less than 1 μM. D Binds to CD16b(NA2).

[0186] The antibodies of the invention further bind to human C1q. Preferably, the antibodies of the invention bind to human C1q at least as strongly as infliximab binds to human C1q.

[0187] The antibodies of the present invention further have complement-dependent cytotoxicity (CDC) of rabbit complement.

[0188] The antibodies of the present invention are further capable of inducing CD14 + CD206 + The induction level is preferably comparable to, equal to, or greater than that of infliximab.

[0189] The antibodies of the present invention are further capable of inhibiting T cell proliferation. The degree of inhibition of T cell proliferation is preferably comparable to, equal to, or greater than the degree of inhibition of T cell proliferation by infliximab.

[0190] Pharmaceutical compositions and treatments

[0191] Treatment of a disease encompasses treating a patient already diagnosed with any form of the disease at any clinical stage or clinical manifestation; delaying the onset or progression or worsening or aggravation of symptoms or signs of the disease; and / or preventing and / or reducing the severity of the disease.

[0192] 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 certain aspects, the human is an adult patient.

[0193] Compositions are described herein that include an anti-TNFα antibody and optionally one or more additional therapeutic agents, such as a second therapeutic agent described below. The composition is typically supplied as part of a sterile pharmaceutical composition that includes a pharmaceutically acceptable carrier. This composition can be in any suitable form (depending on the desired method of administering it to a patient).

[0194] Anti-TNFα antibodies can be administered to patients by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intrathecal, regional or local (e.g., mucosal). The most suitable route of administration in any given case will depend 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.

[0195] In a particularly preferred embodiment, the antibody of the invention is administered orally. If administration is via the oral route, the antibody is preferably IgG, most preferably IgG1.

[0196] In a typical embodiment, 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, antibody concentrations suitable for use in the compositions and methods described herein include, but are not limited to, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, or a concentration within a range between any of the foregoing values, e.g., 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, or 10 mg / kg to 18 mg / kg.

[0197] Depending on the condition being treated, the route of administration, and the age, weight, and condition of the subject, an effective dose of an anti-TNFα antibody may be in the range of about 0.001 to about 750 mg / kg per single (e.g., bolus) administration, multiple administrations, or continuous administration, or to achieve 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. For oral administration, serum concentrations may be extremely low or 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 / kg body weight or about 3 mg to about 30 mg / kg body weight. The antibody may be formulated as an aqueous solution.

[0198] In a particularly preferred embodiment, the antibody of the present invention is administered orally. If administration is via an oral route, the antibody is preferably IgG, most preferably IgG1. If the antibody is administered orally, the daily dose of the antibody is generally in the range of 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. Generally speaking, a favorable dosage is 1 to 200 mg / day, preferably 5 to 100 or 10 to 50 mg / day.

[0199] The pharmaceutical composition may conveniently be presented in unit dosage form containing a predetermined amount of anti-TNFα antibody per dose. Such units 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. Pharmaceutically acceptable carriers may take a wide range of forms, depending, for example, on the condition to be treated or the route of administration.

[0200] Determining the effective dose, total number of doses, and duration of treatment of an anti-TNFα antibody is well within the capabilities of those skilled in the art and can be determined using standard dose escalation studies.

[0201] Therapeutic formulations of anti-TNFα antibodies suitable for use in the methods described herein can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (each referred to herein as "carriers") commonly employed in the art (i.e., buffers, stabilizers, preservatives, isotonicity regulators, nonionic detergents, antioxidants, and other various additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol ed. 1980). Such additives must be nontoxic to the recipient at the dosages and concentrations employed.

[0202] Buffer helps to maintain pH in the range of approaching physiological conditions. Buffer can exist with the concentration of about 2mM to about 50mM scope.Suitable buffer includes organic and inorganic acid and salt thereof, such as citrate buffer (for example, monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture etc.), citrate-phosphate buffer, succinate buffer (for example, succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-sodium succinate mixture etc.), tartrate buffer (for example, tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.), fumarate buffer (for example, fumaric acid-fumaric acid In some embodiments, the present invention provides the present invention with the buffer of at least one embodiment of the present invention. For example, a monosodium mixture, a fumaric acid-disodium fumarate mixture, a monosodium fumarate-disodium fumarate mixture, etc.), a gluconate buffer (e.g., a gluconate-sodium gluconate mixture, a gluconate-sodium hydroxide mixture, a gluconate-potassium gluconate mixture, etc.), an oxalate buffer (e.g., an oxalic acid-sodium oxalate mixture, an oxalic acid-sodium hydroxide mixture, an oxalic acid-potassium oxalate mixture, etc.), a lactate buffer (e.g., a lactic acid-sodium lactate mixture, a lactic acid-sodium hydroxide mixture, a lactic acid-potassium lactate mixture, etc.), and an acetate buffer (e.g., an acetic acid-sodium acetate mixture, an acetic acid-sodium hydroxide mixture, etc.). In addition, a phosphate buffer, a histidine buffer, and a trimethylamine salt such as Tris can be used.

[0203] The pharmaceutical composition of the present invention may further comprise at least one salt, such as sodium chloride. The salt concentration is preferably in the range of 100 mM to 200 mM, such as about 150 mM.

[0204] Preservatives can be added to delay microbial growth, and can be added in an amount within the range of 0.2%-1% (w / v). Suitable preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide and iodide), hexamethylammonium chloride and alkylparabens, such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonic regulators (sometimes referred to as "stabilizers") can be added to ensure the isotonicity of the liquid composition and the isotonic regulators include polyhydroxy sugar alcohols, preferably trihydroxy 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 dissolving therapeutic agents or additives that help prevent denaturation or adhesion to the container wall. Typical stabilizers can be polyhydroxy sugar alcohols (enumerated 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 cyclitols such as inositol, polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, Such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides with 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in the range of 0.1 to 10000 weight per weight of active protein.

[0205] Nonionic surfactants or detergents (also called "wetting agents") may be added to help dissolve the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the protein. Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers ( 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.

[0206] Additional miscellaneous excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), protease inhibitors, and co-solvents.

[0207] The formulations herein may contain a second therapeutic agent in addition to the anti-TNFα antibody thereof. Examples of suitable second therapeutic agents are provided below.

[0208] Depending on a variety of clinical factors, including disease type, disease severity, and patient sensitivity to anti-TNFα antibodies, dosing regimens may vary from once a month to once a day. In a specific embodiment, the anti-TNFα antibody is administered once a day, twice a week, three times a week, every other day, every 5 days, once a week, every 10 days, every two weeks, every three weeks, every four weeks, or once a month, or in any range between any two of the aforementioned values, such as once every four days to once a month, once every 10 days to once every two weeks, or two to three times a week, etc.

[0209] The dosage of the anti-TNFα antibody to be administered will vary depending on the specific antibody, the subject, and 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 route of administration chosen; appropriate dosages can be readily determined by one skilled in the art.

[0210] Those skilled in the art will recognize that the optimal number and interval of individual doses of their anti-TNFα antibody will be determined by the nature and extent of the condition being treated, the form, route and location of administration, and the age and condition of the particular subject being treated, and the physician will ultimately determine the appropriate dose to be administered. This dose may be repeated as many times as necessary. If side effects occur, the amount and / or frequency of the dose may be changed or reduced according to normal clinical practice.

[0211] Condition to be treated

[0212] The present invention relates to a method for treating or preventing a human TNFα-related disease in a subject, the method comprising administering to the subject an antibody as defined herein. The term "TNFα-related disorder" or "TNFα-related disease" refers to the onset, progression or persistence of any disorder, symptom or disease state requiring the involvement of TNFα. Exemplary TNFα-related disorders include, but are not limited to, chronic and / or autoimmune states of generalized inflammation, generalized immune-mediated inflammatory disorders, inflammatory CNS diseases, inflammatory diseases affecting the eyes, joints, skin, mucous membranes, central nervous system, gastrointestinal tract, urinary tract or lungs, states of generalized uveitis, retinitis, HLA-B27+ uveitis, Behcet's disease ( disease), dry eye syndrome, glaucoma, Sjögren's syndrome (syndrome), diabetes (including diabetic neuropathy), insulin resistance, generalized arthritic states, rheumatoid arthritis, osteoarthritis, reactive arthritis and Reiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, amyotrophic lateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidney disease, cystitis, psoriasis (including psoriatic arthritis), hidradenitis suppurativa, panniculitis, pyoderma gangrenosum, SAPHO syndrome (synovitis, acne, impetigo, bone hypertrophy and osteitis), acne, Sweet's sydrome, pemphigus, Crohn's disease (including extraintestinal manifestations), ulcerative colitis, bronchial asthma, hypersensitivity pneumonitis, generalized allergy, allergic rhinitis, allergic sinusitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, Wegener's granulomatosis granulomatosis), Kawasaki syndrome, giant cell arteritis, Churg-Strauss vasculitis, polyarteritis nodosa, burns, graft-versus-host disease, host-versus-graft reaction, rejection events following organ or bone marrow transplantation, systemic and localized states of generalized vasculitis, systemic and cutaneous lupus erythematosus, polymyositis and dermatomyositis, scleroderma, preeclampsia, acute and chronic pancreatitis, viral hepatitis, alcoholic hepatitis, postoperative inflammation (such as after eye surgery (e.g., cataract (lens replacement) or glaucoma surgery)), joint surgery (including arthroscopic surgery), surgery on joint-related structures (e.g., ligaments), oral and / or dental surgery, minimally invasive cardiovascular procedures (e.g.,PTCA, atherectomy, stent placement), laparoscopy and / or endoscopy for intra-abdominal and gynecologic procedures, endoscopic urologic procedures (e.g., prostate surgery, ureteroscopy, cystoscopy, interstitial cystitis), or generalized perioperative inflammation (prophylaxis), bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, pustular dermatitis, cerebral malaria, hemolytic uremic syndrome, allograft rejection, otitis media, snake bites, erythema nodosum, myelodysplastic syndrome, primary sclerosing cholangitis, seronegative spondyloarthropathies, autoimmune hemolytic anemias, orofacial granulomatosis, proliferative purulent stomatitis, aphthous stomatitis, geographic tongue, migratory stomatitis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Bell's palsy palsy, Creutzfeld-Jakob disease, and generalized neurodegenerative disorders.

[0213] 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 effects 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 caused by metastases, dysmenorrhea.

[0214] Specific conditions to be treated include generalized arthritic states, rheumatoid arthritis, osteoarthritis, reactive arthritis, juvenile arthritis; psoriasis, including psoriatic arthritis; inflammatory bowel disease, including Crohn's disease, ulcerative colitis, including proctitis, sigmoiditis, proctosigmoiditis, left-sided colitis, extensive colitis and pancolitis, indeterminate colitis, microscopic colitis, including collagenous and lymphocytic colitis, colitis in connective tissue disease, diversion colitis, colitis in diverticular disease, eosinophilic colitis and pouchitis.

[0215] 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, ileocolonic 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 the localization and disease behavior of any of the above mentioned. Ulcerative colitis can be ulcerative proctitis, proctosigmoiditis, left-sided colitis, ulcerative colitis and pouchitis.

[0216] Combination therapy and other aspects

[0217] Preferably, the patient being treated with their anti-TNFα antibody is also being treated with another conventional agent. For example, patients with inflammatory bowel disease, especially when they have moderate to severe disease, are often also being treated with mesalazine or a derivative or prodrug thereof, a corticosteroid, such as budesonide or prednisolone (oral or intravenous), an immunosuppressant, such as azathioprine / 6-mercaptopurine (6-MP) or methotrexate, cyclosporine, or tacrolimus. Other agents that may be co-administered to the patient include other anti-TNFα antibodies (e.g., infliximab, adalimumab, etanercept, certolizumab pegol, golimumab), integrin antagonists (e.g., natalizumab, vedolizumab), anti-IL-23 antibodies (e.g., MEDI2070), anti-β7 antibodies (e.g., etrolizumab), JAK inhibitors in the JAK / STAT pathway (e.g., tofacitinib), and others. Other agents that may be administered to the patient include immunosuppressants (e.g., azathioprine / 6-MP or methotrexate or oral cyclosporine) in order to maintain stable and longer remission. Yet another aspect of the invention is the use of an anti-TNFα antibody as defined above to reduce inflammation.

[0218] Yet 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 condition.

[0219] Another aspect of the invention is a method of treating an inflammatory condition, the method comprising administering to a patient in need thereof an effective amount of an anti-TNFa antibody as defined above. The inflammatory condition is preferably one of the conditions described above.

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

[0221] Yet another aspect of the present invention is a method for improving the endocytosis of an antibody against TNFα, comprising introducing substitutions E380A and N434A into the amino acid sequence of the antibody so as to obtain a modified antibody with improved endocytosis. The modified antibody is preferably an antibody as described above.

[0222] Yet another aspect of the invention is a method for extending the plasma half-life of an antibody against TNFα, comprising introducing substitutions E380A and N434A into the amino acid sequence of the antibody so as 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 may be increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to the plasma half-life of the unmodified antibody (i.e., the parent antibody lacking the substitutions E380A and N434A).

[0223] Table 1. Overview of sequences in the sequence listing

[0224] SEQ ID NO: Description of amino acid sequence 1 Light chain of the parent antibody Ab-wt used for the modified antibody in the examples 2 The heavy chain of the parent antibody Ab-wt used for the modified antibody 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[Humanized IgG V of Clone 16-22-H05 L > 11 Heavy chain of Ab-AA (based on clone 16-22-H05) 12 Heavy chain of Ab-AA (based on clone 17-22-B03) 13 Fc region of Ab-AA (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 (sc08) L > 22 <![CDATA[V of humanized IgG of Clone 17-22-B03 (sc02) L > 23 Humanized IgG light chain of clone 17-22-B03 (sc08) 24 Light chain of humanized IgG of clone 17-22-B03 (sc02)

[0225] Example

[0226] Antibody variants

[0227] Several variants of anti-TNFα antibodies were generated by introducing substitutions in the Fc region of the antibody amino acid sequence. The mutations were introduced by site-directed mutagenesis using established methods. Briefly, the mutations were introduced by PCR. The forward primer was designed to contain the desired mutation and the reverse primer was designed so that the 5' ends of the two primers were connected back to back (but not overlapping) ( Figure 8 ). The PCR was run for 25 cycles (98°C for 10 s, 64°C for 30 s, 72°C for 3 min). The unmutated PCR template was removed from the PCR product pool using the restriction enzyme DpnI before running the PCR product on an agarose gel. After gel purification of the PCR product, the blunt ends were ligated to obtain a circularized plasmid, which was transformed into competent E. coli cells. After overnight incubation, several clones were picked, plasmid DNA was isolated and sequenced to confirm that the mutation had been incorporated.

[0228] The non-fucosylated variant was generated by adding 0.15 mM of the bait substrate 2-deoxygenase-2-fluoro-2-fucose to the culture medium (Dekkers et al. (2016) Sci Rep 6:36964). This resulted in a significant reduction in the incorporation of fucose into IgG-Fc glycans, as shown in Example 10 below.

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

[0230] name Mutations relative to the parental antibody Ab-wt* None (=parental antibody) Ab-AA** E380A / N434A Ab-AA-2FF** Non-fucosylated variant of Ab-AA

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

[0232] Example 1. Affinity for TNFα

[0233] method:

[0234] The affinity to TNFα is measured by Biacore. The CM5 chip is prepared using standard amine-fixed Biacore procedures. After inserting the CM5 chip, the system is initiated and standardized with BIA standardization solution (Biacore preventive maintenance kit 2). The chip is added to a system with PBS-T running buffer; before fixation, the chip surface is initiated with three injections of 50mM NaOH. Protein A is fixed to the chip surface. For this reason, the protein is diluted to 5 μg / mL in 10mM acetate buffer at pH 4.5 and injected to produce a binding response of about 1000RU in all 4 flow cells. In order to remove non-covalently bound substances from all chip flow cells, three 15 seconds 50mM NaOH washes are performed. On the protein A chip, antibodies are captured in flow cells 2 and 4, and flow cells 1 and 3 are used for reference extraction. The test antibody is diluted to 10nM in PBS-T and 2.5-7.5uL is injected to obtain 120RU of capture antibody. As directed by the supplier, the analyte TNFα was prepared at 500 μg / mL in water and further diluted in the running buffer phosphate buffered saline Tween 20 (PBS-T). Single cycle kinetics were used to evaluate steady-state affinity. For each single cycle analysis cycle, titrations of 5 analyte concentrations were injected over the ligand, and the dissociation of the complex was then measured. The surface was regenerated using glycine pH 1.7. A double reference method was used, in which data from the ligand-bound capture surface (fc 2 and 4) was subtracted from the reference surface without capture ligand (fc 1 and 3, respectively). Blank injections of buffer were performed every 3-4 cycles and then subtracted from the analyte injection cycle to correct for minor changes in the ligand capture surface. Repeated injections of the analyte at the beginning and end of each analytical run were used to check for changes in sample degradation or instrument performance. During the experimental run, all analyses were performed at 25°C and the sample holder was incubated at 10°C. Each experiment was performed at least three times. A 1:1 binding model was used to fit the resulting kinetic data.

[0235] result:

[0236] All antibodies displayed similar binding kinetics to TNFα, indicating that any introduced modifications had not resulted in significant changes in the antigen binding region.

[0237] Table 3: Binding kinetics of human IgG1 variants to TNFα as determined by SPR

[0238]

[0239] Example 2. Efficacy

[0240] method:

[0241] In the presence of serial dilutions of anti-TNFα antibody variants, L929 cells were incubated with 0.25 ng / mL of TNFα and 1 μg / well of actinomycin D. After incubation for 20 h at 37°C / 5% CO2, the proliferative response was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole) and an electron coupling agent (phenazine ethyl sulfate, PES). MTS is converted to a formazan product by dehydrogenases present in metabolically active cells. The amount of formazan product, as measured by absorbance at 492 nM, is proportional to the number of viable cells in the culture.

[0242] result:

[0243] The results are Figure 1 Introducing mutations into the Fc region of anti-TNFα antibodies did not affect potency.

[0244] Example 3. Affinity for Fcγ receptors (CD64, CD32a, CD32b, CD16a, CD16b)

[0245] method:

[0246] The affinity for FcγR is measured by Biacore. The CM5 chip is prepared using a standard amine-fixed Biacore procedure. After inserting the CM5 chip, the system is initiated and standardized with a BIA standardization solution (Biacore preventive maintenance kit 2). The chip is added to a system with PBS-T running buffer; before fixation, the chip surface is initiated with three injections of 50mM NaOH. FcγR is fixed on the chip surface using a His tag capture system. According to the Biacore kit instructions, an anti-His tag chip is prepared using about 12000RU of antibodies deposited on all 4 flow cells. In order to remove non-covalently bound substances from all chip flow cells, three 30-second 10mM glycine pH 1.5 washes are performed. Fcγ receptors are diluted to a range of 0.5-2μg / mL in PBS-T and injected onto the chip using 2.5-5.0μL, thereby producing a capture level between 60 and 200RU. Antibodies are diluted into PBS-T before analysis. Single-cycle kinetics are used to evaluate steady-state affinity. For each single cycle analysis cycle, 5 titration solutions of analyte concentrations were injected over the FcγR ligand, and the dissociation degree of the complex was then measured. The surface was generated using the recommended solution 10mM glycine pH 1.5 to obtain an anti-His capture surface. A double reference method was used, in which data from the ligand-bound capture surface (fc 2 and 4) was subtracted from the reference surface without capture ligand (fc 1 and 3, respectively). Each antibody titration cycle performed a blank injection of buffer and then subtracted from the analyte injection cycle to correct for minor changes in the ligand capture surface. During the experimental run, all analyses were performed at 25°C and the sample rack was incubated at 10°C. Each experiment was performed at least three times.

[0247] result:

[0248] The binding of the engineered anti-TNFα antibody to CD64 was not affected. The introduced mutations did not affect the affinity for CD32a(H), CD32a(R) and CD32b. However, antibody Ab-AA-2FF showed a 4.7-fold increase in affinity for CD16a(V). In particular, the non-fucosylated antibody Ab-AA-2FF also had improved affinity for the low-affinity CD16a receptor and CD16b.

[0249] 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.

[0250]

[0251] Table 5: Affinity for the Fcγ receptors CD16a(V), CD16a(F) and CD16b as determined by SPR. Mean and standard deviation affinities calculated from two or more independent experiments are shown.

[0252]

[0253] Example 4. Affinity for FcRn

[0254] method:

[0255] Using a Biacore 3000 instrument with a CM5 sensor chip coupled to an anti-TNFα IgG1 antibody (about 500 resonance units (RU)), SPR was performed using amine coupling chemicals as described by the manufacturer. Using an amine coupling kit (GE Healthcare), coupling was performed by injecting 2.0ug / mL of each protein into 10mM sodium acetate, pH 4.5. HBS-P buffer pH 7.4 (10mM HEPES, 150mM NaCl, 0.005% surfactant P20) or phosphate buffer pH 6.0 (67nM phosphate buffer, 150mM NaCl, 0.005% Tween 20) was used as running buffer and dilution buffer. Binding kinetics were determined by injecting a titration (1000-31.2nM) of monomeric His-tagged human FcRn (hFcRn) onto fixed antibodies at pH 7.4 or pH 6.0. All SPR experiments were performed at 25°C with a flow rate of 40 ul / min. Binding data were zero adjusted and reference cell values ​​were subtracted. The Langmuir 1:1 ligand binding model provided by BIAevaluation software (version 4.1) was used to determine binding kinetics.

[0256] result:

[0257] The results showed that the wild-type antibody Ab-wt binds to hFcRn with strict pH dependence. 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. All antibody variants show improved binding to FcRn compared to infliximab containing a wild-type IgG1 Fc region.

[0258] Table 6: Affinity of anti-TNFα antibody variants to FcRn at pH 6.0 and pH 7.4 as determined by SPR

[0259]

[0260] NA: Not obtained due to weak binding.

[0261] Example 5. Endocytosis

[0262] method:

[0263] Transwell filters (1.12 cm) with collagen-coated polytetrafluoroethylene (PTFE) membranes with a pore size of 0.4 μm were placed 2 ) were incubated overnight in complete growth medium, and then 1.0x10 6 T84 cells. Transepithelial electrical resistance (TEER) was monitored daily using a MILLICELL-ERS-2 volt-ohm meter. Cultures were grown for 4-5 days before reaching confluence with TEER values ​​of approximately 1000-1300 Ωxcm 2 . Prior to the experiment, the monolayer was starved for 1 h in Hank's balanced salt solution (HBSS). Subsequently, 400 nM of antibody variants or IFX were added to the apical Transwell chamber alone or together with 4000 nM human myeloma IgG with irrelevant specificity. Samples were collected from the basolateral reservoir at 0 and 4 h after addition. The antibody concentration in the basolateral reservoir was determined by ELISA. In brief, 96-well Maxisorp plates were coated overnight with recombinant TNFα or anti-human Fc-specific antibodies from goats, all diluted to 1 μg / ml in PBS. Next, the plates were blocked at room temperature for 2 h with PBS containing 4% skim milk, followed by washing 4 times with PBS containing 0.05% Tween 20. Samples collected during the endocytosis transport experiment were added to the wells and incubated at room temperature for 2 h, followed by washing as above. Captured antibody variants, IFX or total IgG were detected using anti-human Fc-specific antibodies conjugated with alkaline phosphatase (ALP) from goats. Binding was visualized by adding 100 μL ALP substrate and recording the 405 nM absorbance spectrum.The amount of transported antibody variant, IFX and total IgG was calculated from the standard curve for each individual antibody variant.

[0264] Transcytosis of antibody variants across polarized human epithelial cells

[0265] result:

[0266] The engineered anti-TNFα antibody variants were tested for transcytosis across cell monolayers and compared to IFX, another human IgG1 anti-TNFα antibody. Figure 2 Depicted in . The IgG1 antibody Ab-AA with a wt Fc region was transported with approximately 1.8 times higher efficiency compared to IFX. Similar to the transport of Ab-AA, a significant increase was also observed for the non-fucosylated version Ab-AA-2FF.

[0267] Transcytosis of antibody variants across polarized human epithelial cells in the presence of competing IgG

[0268] result:

[0269] When anti-TNFα antibody variants were incubated with a 10-fold excess of human myeloma IgG, the total amount of immunoglobulin transported across polarized T84 cell monolayers from the apical side to the basolateral reservoir was comparable for all antibodies 4 hours after addition. However, the increased affinity for FcRn at pH 6.0 resulted in a significantly higher percentage of specific anti-TNFα transport across the cell monolayer also in the presence of excess competing human IgG of irrelevant specificity. Figure 3 Depicted in.

[0270] Example 6. ADCC

[0271] method:

[0272] The ADCC reporter gene bioactivity assay core kit from Promega was used. Briefly, 1x10 5 mTNFα CHO-K1 target cells at 100 μL / mL were seeded into white (clear bottom) tissue culture plates. The plates were incubated overnight at 37°C and 5% CO2. On day 2, 95 μL of the assay medium was removed and 3x10 6 The plates were then incubated at 37°C / 5% CO2 for 6 h. Near the end of the incubation, BioGlo TM The plate was allowed to equilibrate to room temperature for 10-20 min, after which 75 μL of BioGlo was added to each well. TM Reagents. After incubation in the dark for 5-10 min, fluorescence was measured. A 4-PL model was used to fit the data.

[0273] result:

[0274] Results (see Figure 4 ) showed that all anti-TNFα antibodies induced ADCC, but with varying strengths. Compared to the wild-type antibody Ab-wt, Ab-AA showed similar ADCC activity, while the non-fucosylated antibody variant Ab-AA-2FF had significantly improved ADCC.

[0275] Example 7. C1q Binding

[0276] method:

[0277] ELISA was performed using a 96-well MaxiSorp plate, where the wells were coated with human TNFα diluted to 1 μg / mL in PBS. After incubation overnight at 4°C, the plate was blocked for 1 h with PBS containing 4% skim milk and washed 4 times with PBS containing 0.05% Tween 20 (PBS-T). Subsequently, a titrated amount of anti-TNFα IgG antibody was diluted in PBS-T, added and incubated for 1 h at room temperature. After washing with PBS-T, human C1q (0.5 μg / mL) was diluted in 0.1M Veronal buffer (0.25mM CaCl2 and 0.8mM MgCl2 pH 7.2), added to the wells and incubated for 1 h. Next, the wells were washed as above, after which rabbit anti-human C1q diluted 1:5000 in PBS-T was added to the wells and incubated for 1 h. After washing, HRP-conjugated anti-rabbit IgG from monkey diluted 1:5000 in PBS-T was added. Next, 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.

[0278] result:

[0279] The anti-TNFα antibody variants were captured onto human TNFα followed by the addition of human C1q. Results (see Figure 5 ) shows that the antibodies bind to C1q, but with varying strengths. The ranking from strongest to weakest binding is as follows:

[0280] Ab-AA=IFX>Ab-AA-2FF.

[0281] Example 8. Induction of regulatory macrophages

[0282] method:

[0283] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy buffy coats. Cells were separated by Ficoll gradient centrifugation. Cells from two individual donors were mixed in equal numbers and 2×10 of the mixture was added in a total volume of 100 μL / well. 5Cells were seeded into 96-well plates. The plates were incubated at 37°C / 5% CO2 for 48h. After 48h, anti-TNFα antibody variants or IFX were added to reach a final concentration of 10μg / mL. Each compound was added in five or six replicates. The final volume was 150μL / well. Human serum IgG1 (Sigma#I5154) was used as a control. After adding the compound, the mixed lymphocyte reaction (MLR) was cultured for another 2 days at 37°C / 5% CO4. Afterwards, the plates were washed with PBS / 5mM EDTA (PBS / EDTA) and the plates were incubated at room temperature with 50μL / well PBS / EDTA for 20min. The plates were centrifuged and the liquid was discarded. The antibodies were diluted in PBS / EDTA (anti-CD14-PE, anti-CD206-APC, all diluted 1:10). The cells were resuspended in 50μL antibody solution and incubated at room temperature for 20min. Afterwards, the cells were washed with PBS / EDTA and resuspended in 50 μL PBS / EDTA. The stained samples were analyzed on a FACSFortessa using FACSDiva software. FlowJo software was used for analysis.

[0284] result:

[0285] Induction of regulatory macrophages was analyzed in four independent MLRs and was successful in all experiments (IFX was compared to IgG control). Figure 6 The induction level of IFX may vary between experiments due to the fact that each experiment was performed using different donors with inter-individual variability. All tested anti-TNFα antibody variants induced CD14 with slight variability between compounds. + CD206 + Regulatory macrophages. Ab-AA-2FF induced more regulatory macrophages than IFX.

[0286] Example 9. Inhibition of T cell proliferation

[0287] method:

[0288] PBMCs were isolated from healthy buffy coats. Cells were separated by Ficoll gradient centrifugation. Cells from two individual donors were mixed in equal numbers and 2×10 of the mixture was added in a total volume of 100 μL / well. 5Cells were seeded into 96-well plates. The plates were incubated at 37°C / 5% CO2 for 48 h. After 48 h, anti-TNFα antibody variants or IFX were added to reach a final concentration of 10 μg / mL. Each compound was added in five or six replicates. The final volume was 150 μL / well. Human serum IgG1 (Sigma #I5154) was used as a control. After adding the compounds, the mixed lymphocyte reaction (MLR) was incubated at 37°C / 5% CO2 for another 2 days. Afterwards, titrated thymidine ( 3 H-thymidine, 0.5 microcurie / well) was added to the culture. The culture was incubated at 37°C / 5% CO2 for 18 h. Samples were harvested using a Microbeta Filtermat 96-well harvester and analyzed using a Microbeta microplate counter equipped with a single detector. Samples were counted for 10 seconds / well and converted to counts per minute (cpm).

[0289] result:

[0290] Inhibition of T cell proliferation was measured in three independent MLRs and was defined as successful if IFX, a positive control, induced inhibition. The level of inhibition by IFX may vary between experiments, presumably due to differences in 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-AA-2FF showed significantly enhanced inhibition compared to IFX, while Ab-AA showed significantly less inhibition of T cell proliferation compared to IFX (see Figure 7 ).

[0291] Embodiment 10. Analysis of N-glycans

[0292] method:

[0293] Each IgG variant (1mg / ml) of 50 μ L is spun down for 10min at 13000 × g, then 1 μ g trypsin dissolved in 100 μ L 50mM ammonium bicarbonate (pH 7.8) is added and incubated overnight at 37 ° C. Centrifugal device is spun down for 10min at 13000 × g, and the flow-through is transferred to Eppendorf tube (Eppendorf tube) and dried in SpeedVac (HetoMaxi dryer). The dried sample is dissolved in 20 μ L 1% formic acid, ultrasonically treated for 30s, and centrifuged for 10min at 16100 × g. Then, each sample is transferred to a new vial, and reversed phase (C18) nano online liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of proteolytic peptides is performed using Dionex Ultimate3000UHPLC system (Thermo Fisher 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 backwash 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% aqueous acetonitrile for 60 minutes at a flow rate of 0.3 μL / min. The LC system was coupled to a Q exactive hybrid quadrupole orbital trap 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 at a standardized collision energy of 20 to obtain an orbital trap full spectrum scan in the mass range of m / z 300-2000, and then MS / MS was performed on the ten most intense ions in the orbital trap.

[0294] Data analysis was performed on Xcalibur v2.0. The MS / MS spectra of all N-glycopeptides were extracted by oxonium ion search; 204.086 (N-acetylhexosamine) and 366.1388 (N-acetylhexosamine-hexose). By using the HCD fragmentation at 20° of standardized collision energy, the glycan structure and peptide mass corresponding to IgG were detected. The extracted ion chromatogram of the target ethylene glycol-peptide (EEQYNSTYR for IgG1) was extracted with an accuracy of 10ppm, and the corresponding MS / MS spectra were manually verified. The HCD fragmentation at 35° of standardized collision energy was used to detect the peptide sequence and verify that the peptide mass corresponds to the correct peptide sequence. The area under the curve of all extracted ethylene glycol-peptides was calculated and the percentage ratio of each glycoform was determined.

[0295] result:

[0296] For Ab-AA, 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 predominant N-glycan forms contained core fucose. To prepare a "non-fucosylated" version of Ab-AA (Ab-AA-2FF), the bait substrate 2-deoxy-2-fluoro-1-fucose (2FF) was used. MS imaging of this antibody revealed that this strategy successfully resulted in a significant reduction in the incorporation of fucose, as a drop from >90% to <10% was detected. The predominant N-glycan forms after treatment were the same as the variants prepared in the absence of 2FF, but these structures lacked fucose (see also Fig. 9 ).

[0297] Table 7: Percentage of N-glycan forms attached to N297 of anti-TNFα IgG antibodies

[0298]

Claims

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 500 nM, and it further has no affinity or low affinity for human FcRn at pH 7.4, the low affinity being characterized by a K D Greater than 10 μM, wherein the amino acid sequence of the antibody comprises amino acids 380A and 434A according to EU numbering, and wherein the antibody comprises: V L domain, the V L The structural domain comprises 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 domain, the V H The domain comprises 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, wherein the amino acid sequence of the antibody further comprises amino acid 307T according to EU numbering.

3. 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.

4. The antibody of claim 1, wherein the high affinity for human FcRn is characterized by a dissociation equilibrium constant (K D ) is less than 300nM.

5. The antibody of claim 1, wherein the antibody has a K of less than 100 pM. D Binds to human TNFα.

6. The antibody of claim 1, which is transported across polarized cell monolayers from the apical side to the basolateral side in an amount greater than infliximab.

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

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

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

20. H A domain and a V domain having an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22 L Structural domain. The antibody according to claim 10 , comprising a light chain having an amino acid sequence as shown in SEQ ID NO: 23 or SEQ ID NO: 24 and a heavy chain having an amino acid sequence as shown in SEQ ID NO:

12.

12. The antibody of claim 1, wherein the antibody specifically binds to human TNFα.

13. A nucleic acid encoding the antibody according to any one of claims 1 to 12.

14. The antibody according to any one of claims 1 to 12 for use in treating an inflammatory condition.

15. The antibody for use according to claim 14, wherein the inflammatory condition is an inflammatory disorder of the gastrointestinal tract.

16. The antibody for use according to claim 14, wherein the treatment comprises oral administration of an effective amount of the antibody.

17. The antibody for use according to claim 14, wherein the antibody is administered topically.

18. The antibody for use according to claim 15, wherein the antibody is administered topically.

19. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 12.

20. A method for improving the endocytic transport of an antibody as defined in claim 1, said method comprising introducing the substitutions E380A and N434A according to EU numbering into the amino acid sequence of the Fc region of said antibody.

21. A method for extending the plasma half-life of an antibody as defined in claim 1, said method comprising introducing the substitutions E380A and N434A according to EU numbering into the amino acid sequence of the Fc region of said antibody.

22. Use of an antibody according to any one of claims 1 to 12 in the preparation of a medicament for treating TNFa-associated inflammatory conditions.

23. The use according to claim 22, wherein the TNFα-related inflammatory condition is an inflammatory disorder of the gastrointestinal tract.

24. The use according to claim 22, wherein the treatment comprises oral administration of an effective amount of the antibody.

25. The use according to claim 22, wherein the antibody is administered topically.

26. The use according to claim 23, wherein the antibody is administered topically.

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