Antibody variants

Abstract

To provide antibodies which bind to TNFα and comprise a modified Fc region.SOLUTION: An antibody of the invention comprises a TNFα-binding domain and an FcRn binding site, the antibody comprising a specific amino acid sequence. The amino acid sequence of the antibody has improved resistance against proteolytic degradation and good effector functions and / or pharmacokinetic properties.SELECTED DRAWING: None

Description

[Technical field]

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

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

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

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

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

[0006] WO 2012 / 087746A1 and Kinder et al. (2013) The Journal Of Biological Chemistry Vol. 288, No. 43, pp. 30843-30854 investigated various mutations in antibody Fc regions to improve resistance to proteolysis.

[0007] There is an ongoing need for antibodies with improved effector functions, pharmacokinetics and / or resistance to proteolysis. Summary of the Invention

[0008] The inventors of the present application have found that certain mutations in the antibody Fc region confer favorable properties to the antibody, including improved proteolytic resistance and increased affinity for FcRn at pH 6. The antibodies with the mutations have improved pharmacokinetic properties. Furthermore, the antibodies exhibit superior effector functions compared to unmodified antibodies and / or known antibodies such as infliximab (IFX).

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

[0100] : [1] An antibody comprising a TNFα-binding domain and an FcRn-binding site, the antibody having an amino acid sequence: (i) deletions of amino acids 233P, 234V, 235A, and at amino acid position 236; and (ii) amino acid 434A or amino acids 252Y, 254T, and 256E An antibody comprising: [2] The antibody according to item [1], which is a modified antibody having the substitutions E233P, L234V and L235A, and further having a deletion of G236. [3] The antibody according to item [2], further comprising the substitution N434A. [4] The antibody according to item [2], wherein the antibody further has substitutions M252Y, S254T and T256E. [5] The antibody according to any one of items [1] to [4], wherein the amino acid sequence of the antibody further comprises amino acids 239D, 330L and 332E. [6] The antibody according to item [5], wherein the antibody is a modified antibody having substitutions S239D, A330L and I332E. [7] The antibody according to item [1], wherein the amino acid sequence of the antibody comprises amino acids 233P, 234V, 235A, 239D, 330L, 332E and 434A, and comprises a deletion at amino acid position 236. [8] The antibody according to item [7], which is a modified antibody having the substitutions E233P, L234V, L235A, S239D, A330L, I332E and N434A, and further having a deletion of G236. [9] The antibody according to item [7] or [8], comprising the amino acid sequence shown in SEQ ID NO: 29.

[10] The antibody according to item [1], wherein the amino acid sequence of the antibody comprises amino acids 233P, 234V, 235A, 239D, 330L, 332E, 252Y, 254T and 256E, and comprises a deletion at amino acid position 236.

[11] The antibody of item

[10] , wherein the antibody is a modified antibody having the substitutions E233P, L234V, L235A, S239D, A330L, I332E, M252Y, S254T and T256E, and further having a deletion of G236.

[12] The antibody according to item

[10] or

[11] , comprising the amino acid sequence shown in SEQ ID NO: 28.

[13] The antibody according to item [1], wherein the amino acid sequence of the antibody comprises amino acids 233P, 234V, 235A, 326A, 332E, 333A and 434A, and comprises a deletion at amino acid position 236.

[14] The antibody of item

[13] , wherein the antibody is a modified antibody having the substitutions E233P, L234V, L235A, K326A, I332E, E333A and N434A, and further having a deletion of G236.

[15] The antibody according to item

[13] or

[14] , comprising the amino acid sequence shown in SEQ ID NO: 30.

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

[15] , which has an affinity for human FcRn at pH 6 greater than the affinity of infliximab.

[17] Dissociation constant K<500 nM DThe antibody according to any one of items [1] to

[16] , which has affinity for human FcRn at pH 6.

[18] Dissociation constant K<400 nM D The antibody according to any one of items [1] to

[17] , which has affinity for human FcRn at pH 6.

[19] Dissociation constant K<300 nM D The antibody according to any one of items [1] to

[18] , which has affinity for human FcRn at pH 6.

[20] Dissociation constant K<200 nM D The antibody according to any one of items [1] to

[19] , which has affinity for human FcRn at pH 6.

[21] A dissociation constant K in the range of 5 nM to 500 nM, or 10 nM to 400 nM, or 25 nM to 300 nM, or 50 nM to 200 nM, or 75 nM to 175 nM. D The antibody according to any one of items [1] to

[20] , which has affinity for human FcRn at pH 6.

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

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

[23] A dissociation constant K of greater than 10 μM D The antibody according to any one of items [1] to

[22] , which has affinity for human FcRn at pH 7.4.

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

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

[25] The affinity for human FcRn at pH 7.4 was characterized by its K D The antibody according to any one of items [1] to

[22] , wherein the value is so low that it cannot be measured by SPR.

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

[25] , which binds to

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

[26] , which binds to

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

[27] , which binds to

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

[28] , which binds to

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

[29] , which binds to

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

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

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

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

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

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

[34] The antibody of item

[32] or

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

[35] The antibody according to any one of items

[31] to

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

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

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

[37] The antibody of item

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

[38] The antibody according to any one of items

[31] to

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

[39] CD16a(V) has a K of less than 500 nM, or less than 300 nM, or less than 200 nM, or less than 100 nM D The antibody according to any one of items [1] to

[38] , which binds to

[40] CD16a(F) has a K value of less than 10 μM or less than 1 μM D The antibody according to any one of items [1] to

[39] , which binds to

[41] K for CD16b(NA2) is less than 10 μM, or less than 5 μM, or less than 1 μM D The antibody according to any one of items [1] to

[40] , which binds to

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

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

[43] CD14 + CD206 + The antibody according to any one of items [1] to

[42] , which is capable of inducing macrophages.

[44] CD14 at levels higher than infliximab + CD206 + The antibody according to any one of items [1] to

[43] , which is capable of inducing macrophages.

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

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

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

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

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

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

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

[47] , comprising a domain.

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

[48] , comprising a domain.

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

[49] , comprising a light chain having the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

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

[47] , comprising a domain.

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

[51] , comprising a domain.

[53] The antibody according to item

[51] or

[52] , comprising a light chain having the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27.

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

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

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

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

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

[55] .

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

[56] , which binds to

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

[57] , which binds to

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

[58] , wherein the ratio is a value (ng / mL) relative to the antibody titer.

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

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

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

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

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

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

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

[62] , which is capable of blocking the interaction between human TNFα and TNF receptor I (TNFRI).

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

[63] , which is capable of blocking the interaction between human TNFα and TNF receptor II (TNFRII).

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

[64] , which is capable of suppressing cell proliferation of peripheral blood mononuclear cells in a mixed lymphocyte reaction.

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

[65] , which is capable of suppressing LPS-induced secretion of interleukin-1β derived from monocytes.

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

[66] , wherein the antibody has a titer of less than 1 nM.

[68] On a molar basis, IC for inhibiting LPS-induced secretion of interleukin-1β 50 Values ​​are IC 50 The antibody according to item

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

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

[68] , which is capable of suppressing LPS-induced secretion of TNFα derived from monocytes.

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

[69] , wherein the antibody has a titer of less than 1 nM.

[71] On a molar basis, IC for inhibiting LPS-induced secretion of TNFα 50 Values ​​are IC 50 The antibody according to item

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

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

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

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

[72] , which is more resistant to proteolysis than a wild-type antibody.

[74] The antibody according to item

[73] , wherein the wild-type antibody is infliximab.

[75] The antibody according to item

[73] , wherein the wild-type antibody differs from the antibody only in that the Fc region of the wild-type antibody has only wild-type amino acids.

[76] The antibody described in

[73] , wherein the wild-type 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.

[77] The antibody according to any one of items

[73] to

[76] , wherein the proteolysis includes degradation by matrix metalloproteinase 3 (MMP-3).

[78] The antibody according to any one of items

[73] to

[77] , wherein the proteolysis includes proteolysis by immunoglobulin G-degrading enzyme (IdeS) of Streptococcus pyogenes.

[79] The antibody according to any one of items

[73] to

[78] , wherein the proteolysis includes degradation by endoprotease Glu-C (Gluc) derived from Staphylococcus aureus strain V8.

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

[79] .

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

[80] .

[82] A cell containing the nucleic acid of item

[80] or the vector or plasmid of item

[81] .

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

[79] , comprising culturing the cell according to item

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

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

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

[85] The antibody for use according to item

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

[86] The antibody for use according to item

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

[87] The antibody for use according to item

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

[88] The antibody for use according to item

[86] or

[87] , wherein the inflammatory disease of the digestive tract is Crohn's disease.

[89] The antibody for use according to item

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

[90] The antibody for use according to item

[86] or

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

[91] The antibody for use according to item

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

[92] The antibody for use according to item

[86] or

[87] , wherein the inflammatory disease of the digestive tract is microscopic colitis.

[93] The antibody for use according to item

[84] , wherein the inflammatory disease is arthritis.

[94] The antibody for use according to item

[84] or

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

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

[84] to

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

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

[84] to

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

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

[79] .

[98] A method for improving transcytosis of an antibody against TNFα, comprising introducing the substitutions E233P, L234V and L235A, deleting G236, and further substitutions (a) or (b) below: (a) M252Y, S254T and T256E, (b) N434A, and, optionally, further introducing one or more other substitutions described herein.

[99] A method for increasing the plasma half-life of an antibody against TNFα, comprising introducing the substitutions E233P, L234V and L235A, deleting G236, and the further substitutions (a) or (b) below: (a) M252Y, S254T and T256E, (b) N434A, and, optionally, further introducing one or more other substitutions described herein.

[0100] A method for improving the resistance to proteolysis of an antibody against TNFα, comprising introducing the substitutions E233P, L234V and L235A, deleting G236 and further substitutions (a) or (b) below: (a) M252Y, S254T and T256E, (b) N434A, and, optionally, further introducing one or more other substitutions described herein. [Brief description of the drawings]

[0010] [Figure 1] Potency of anti-TNFα antibody variants to neutralize human TNFα in the L929 assay. Dose-response curves of the TNFα antibody variants and reference infliximab are shown. [Diagram 2] Transport of anti-TNFα IgG variants across polarized T84 cells. Amounts of anti-TNFα antibody variants and infliximab (IFX) from the apical to the basolateral reservoir 4 h after addition. Expressed as ng / cm2. Error bars indicate SD of two to four individual monolayers. [Diagram 3]Transport of anti-TNFα IgG variants across polarized T84 cells in the presence of excess myeloma IgG. Amounts of anti-TNFα antibody IFX and antibody variants transported from the apical to the basolateral reservoir in the presence of a 10-fold excess of human myeloma IgG 4 hours after addition. Expressed as ng / cm2. Error bars represent SD of 3-4 individual monolayers. [Figure 4] ADCC activity. Induction of ADCC by anti-TNFα antibody variants and Ab-wt. [Diagram 5] Induction of CD14+CD206+ macrophages by each compound compared to that by IFX. Data pooled from four independent experiments. Bars represent the mean and error bars represent SEM. [Figure 6] Inhibition of T cell proliferation by each compound compared to IFX. Data pooled from three independent experiments. Bars represent the mean and error bars represent SEM. [Figure 7] Resistance to proteolysis by MMP-3. [Figure 8] Resistance to proteolysis by IdeS. [Figure 9] Resistance to proteolysis by Gluc. [Figure 10] Schematic representation of site-directed mutagenesis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention relates to an antibody capable of binding to TNFα and comprising a modified Fc region. The antibody has improved proteolytic resistance. The antibody further has high affinity for human FcRn at pH 6 and low affinity for human FcRn at pH 7.4. The amino acid sequence of the antibody comprises amino acids 233P, 234V, 235A and comprises a deletion at amino acid position 236 (EU numbering). The antibody further comprises amino acid 434A or amino acids 252Y, 254T and 256E (EU numbering).

[0012] Throughout this specification, when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is typically used (Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is typically used (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference). Unless otherwise specified, reference to residue numbers in the variable domain of an antibody refers to residue numbering according to the Kabat numbering system. Unless otherwise specified, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system (see, for example, WO 2006 / 073941).

[0013] antibody In the present application, the term "antibody" is used as a synonym for "immunoglobulin" (Ig), which is defined as a protein including all conventionally known antibodies and their functional fragments belonging to the IgG, IgM, IgE, IgA, or IgD classes (or any subclass thereof). In the present invention, an "functional fragment" of an antibody / immunoglobulin is defined as an antigen-binding fragment or other derivative of a parent antibody that essentially maintains one or more properties of such parent antibody. An "antigen-binding fragment" or "antigen-binding domain" of an antibody / immunoglobulin is defined as a fragment that retains the antigen-binding region (e.g., the variable region of an IgG). The "antigen-binding region" of an antibody is typically found in one or more hypervariable regions of the antibody, i.e., the CDR-1, -2 and / or -3 regions. The antibody of the present invention may be part of a bifunctional or multifunctional construct.

[0014] Preferably, the antibody is a monoclonal antibody. As used herein, the term "monoclonal antibody" is not limited to the antibody produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody that originates from a single clone, including any eukaryotic, prokaryotic or phage clone, and not the method by which it is produced. Monoclonal antibody can be produced using a wide variety of techniques known in the art, such as using hybridoma, recombinant and phage display technology, or a combination thereof (Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor NY).

[0015] In other embodiments, including those directed to in vivo use of anti-TNFα antibodies in humans, chimeric, primatized, humanized, or human antibodies can be used. In preferred embodiments, the antibody is a human or humanized antibody, more preferably a monoclonal human or humanized antibody.

[0016] In another specific embodiment, the antibody of the present invention is an immunoglobulin, preferably an immunoglobulin G (IgG). The subclasses of the IgG of the present invention include, but are not limited to, IgG1, IgG2, IgG3, and IgG4. Preferably, the IgG of the present invention is subclass 1, 2, or 4, i.e., IgG1, IgG2, or IgG4 molecule, respectively. Most preferably, the IgG of the present invention is subclass 1, i.e., IgG1 molecule.

[0017] TNFα binding domain The TNFα-binding domain of the antibody of the present invention is not particularly limited. It can be derived from any antibody capable of binding to TNFα.

[0018] Preferably, the antibody of the present invention specifically binds to TNFα. As used herein, an antibody "specifically recognizes" or "specifically binds" to human TNFα if it can distinguish between human TNFα and one or more reference molecules. Preferably, the IC for binding to each of the reference molecules is 0.01 to 0.05. 50 Values ​​are IC for binding to TNFα 50 At least 1,000 times greater than the value. In its most common form (and when no defined reference is given), "specific binding" refers to the ability of an antibody to distinguish between human TNFα and unrelated biomolecules, for example, as measured by specificity assay methods known in the art. Such methods include, but are not limited to, Western blots and ELISA tests. For example, a standard ELISA assay can be performed. Typically, the determination of binding specificity is performed by using a set of about 3-5 unrelated biomolecules, such as milk powder, BSA, transferrin, etc., rather than a single reference biomolecule. In one embodiment, specific binding refers to the ability of an antibody to distinguish between human TNFα and human TNFβ.

[0019] The antibody of the present invention is L Domains and V H Includes domain. V LA domain includes a CDR1 region (CDRL1), a CDR2 region (CDRL2), a CDR3 region (CDRL3), and a framework region. H A domain comprises a CDR1 region (CDRH1), a CDR2 region (CDRH2), a CDR3 region (CDRH3), and framework regions.

[0020] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs was provided by Kabat EA et al. (1991) (Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, Kabat's CDR definition applies only to CDR1, CDR2 and CDR3 (CDRL1, CDRL2, CDRL3, or L1, L2, L3) of the light chain variable domain and CDR2 and CDR3 (CDRH2, CDRH3, or H2, H3) of the heavy chain variable domain. However, as used herein, CDR1 (CDRH1 or H1) of the heavy chain variable domain is defined by the following residues (Kabat numbering): it starts at position 26 and ends before position 36.

[0021] In certain embodiments, the antibody of the invention comprises: (i) a CDR1 region having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:4, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:5; L and (ii) a V domain comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:6, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:7, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:8. H Includes the domain.

[0022] In a more preferred embodiment, the antibody of the present invention has the amino acid sequence shown in SEQ ID NO:9. HIn another more preferred embodiment, the antibody comprises a V domain having the amino acid sequence set forth in SEQ ID NO:10. L Most preferably, the antibody of the invention comprises (i) a V domain having the amino acid sequence set forth in SEQ ID NO:9. H domain, and (ii) a V domain having the amino acid sequence set forth in SEQ ID NO:10. L Includes the domain.

[0023] In another specific embodiment, the antibody of the invention comprises: (i) a CDR1 region having the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 region having the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 16. L and (ii) a V domain comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:17, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:18, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:19. H Includes the domain.

[0024] In a more preferred embodiment, the antibody of the present invention has the amino acid sequence shown in SEQ ID NO: 20. H In another more preferred embodiment, the antibody comprises a V domain having the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:22. L Most preferably, the antibody of the invention comprises (i) a V domain having the amino acid sequence set forth in SEQ ID NO:21. H domain, and (ii) a V domain having an amino acid sequence as set forth in SEQ ID NO:21 or SEQ ID NO:22. L Includes the domain.

[0025] The antibody of the present invention has high affinity for human TNFα. D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present invention have a dissociation equilibrium constant of about 2x10 as measured using surface plasmon resonance (SPR) technology on a BIACORE instrument. -10 Less than M, preferably 1.5x10 -10 Less than M, preferably 1.25x10 -10Less than M, more preferably 1x10 -10 Less than M, most preferably 7.5x10 -11 Less than M or 5x10 -11 The dissociation equilibrium constant (K D ) binds to human TNFα. D Measurements are performed as described in Example 1.

[0026] Fc Region Modification An "altered Fc region" comprises an amino acid sequence that differs from a native sequence Fc region due to at least one "amino acid modification" or "mutation" as defined herein. Preferably, the modified Fc region comprises a modified FcRn-binding site having at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, and preferably about 1 to about 5 amino acid substitutions, in the native sequence FcRn-binding site or in the parent antibody FcRn-binding site, compared to the native sequence FcRn-binding site or compared to the parent antibody FcRn-binding site. Alternatively, the antibody may have modifications outside the FcRn-binding site that affect affinity to FcRn, e.g., by structural changes. Usually, affinity to human FcRn at pH 6 is increased by the modification. Preferably, affinity to human FcRn at pH 7.4 is not substantially affected by the modification. The modifications can be produced by methods known per se, for example by site-directed mutagenesis as described in "Antibody Engineering-Methods and Protocols", edited by Patrick Chames, 2nd ed., 2012, Chapter 31 (ISBN 978-1-61779-973-0).

[0027] The amino acid sequence of the antibody of the invention comprises the amino acid proline at position 233, the amino acid valine at position 234 and the amino acid alanine at position 235, and further has a deletion of the amino acid at position 236 (EU numbering). This is referred to herein as "233P / 234V / 235A / 236del". The native amino acid at position 233 of an unmodified human IgG antibody is glutamic acid (E). The native amino acid at position 234 of an unmodified human IgG antibody is leucine (L). The native amino acid at position 235 of an unmodified human IgG antibody is leucine (L). The native amino acid at position 236 of an unmodified human IgG antibody is glycine (G). Thus, the antibody of the invention can be obtained by introducing the mutations E233P, L234V, L235A and G236del into the antibody. This is referred to herein as "E233P / L234V / L235A / G236del". Preferably, the antibody of the invention is obtainable or obtained by substitution of glutamic acid with proline at position 233, substitution of leucine with valine at position 234, substitution of leucine with alanine at position 235, and deletion of glycine at position 236.

[0028] The amino acid sequence of the antibody of the invention further comprises (i) the amino acid alanine at position 434 or (ii) the amino acid tyrosine at position 252, the amino acid threonine at position 254 and the amino acid glutamic acid at position 256, referred to herein as 434A and 252Y / 254T / 256E, respectively. The native amino acid at position 434 of an unmodified human IgG antibody is asparagine (N). The native amino acid at position 252 of an unmodified human IgG antibody is methionine (M). The native amino acid at position 254 of an unmodified human IgG antibody is serine (S). The native amino acid at position 256 of an unmodified human IgG antibody is threonine (T). Thus, the antibody of the invention can be obtained by introducing the further mutations N434A or M252Y / S254T / T256E into the antibody.

[0029] That is, the amino acid sequence of the antibody of the present invention comprises 233P / 234V / 235A / 236del / 434A or 233P / 234V / 235A / 236del / 252Y / 254T / 256E, which can be obtained by introducing the mutations E233P / L234V / L235A / G236del / N434A or E233P / L234V / L235A / G236del / M252Y / S254T / T256E into the amino acid sequence of the antibody, for example, into the amino acid sequence of an antibody whose Fc region has a non-modified or wild-type amino acid sequence.

[0030] The remaining amino acid sequence of the Fc region can be the same as the native amino acid sequence of a typical human IgG, but the amino acid sequence of the antibody can contain one or more mutations or substitutions relative to the native amino acid sequence of the Fc region of a native antibody, so long as the antibody still has TNFα-binding activity, FcRn-binding activity at pH 6.0, and one or more effector functions after the modifications described below.

[0031] In a preferred embodiment, the antibody of the invention has at least one, or at least two, or at least three additional substitutions. In one embodiment, the amino acid sequence of the antibody preferably has the amino acids 239D / 330L / 332E, which can be obtained or obtained by introducing the substitutions S239D / A330L / I332E. In another embodiment, the amino acid sequence of the antibody preferably has the amino acids 326A / 332E / 333A, which can be obtained or obtained by introducing the substitutions K326A / I332E / E333A.

[0032] In a preferred embodiment, the amino acid sequence of the antibody of the invention comprises 233P / 234V / 235A / 236del / 239D / 330L / 332E / 434A. This antibody can be obtained by introducing the mutations E233P / L234V / L235A / 236del / S239D / A330L / I332E / N434A into the amino acid sequence of an antibody, for example an antibody whose Fc region has a non-modified or wild-type amino acid sequence.

[0033] In another preferred embodiment, the amino acid sequence of the antibody of the invention comprises 233P / 234V / 235A / 236del / 239D / 330L / 332E / 252Y / 254T / 256E. This antibody can be obtained by introducing the mutations E233P / L234V / L235A / 236del / S239D / A330L / I332E / M252Y / S254T / T256E into the amino acid sequence of the antibody, for example, into the amino acid sequence of an antibody whose Fc region has a non-modified or wild-type amino acid sequence.

[0034] In another preferred embodiment, the amino acid sequence of the antibody of the invention comprises 233P / 234V / 235A / 236del / 326A / 332E / 333A / 434A. This antibody can be obtained by introducing the mutations E233P / L234V / L235A / 236del / K326A / I332E / E333A / N434A into the amino acid sequence of an antibody, for example an antibody whose Fc region has a non-modified or wild-type amino acid sequence.

[0035] In a preferred embodiment, the Fc region of an antibody of the invention, including the hinge region, comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.

[0036] In one embodiment, the heavy chain of the antibody of the invention has the amino acid sequence shown in SEQ ID NO: 2, into which the mutations E233P / L234V / L235A / 236del / S239D / A330L / I332E / N434A have been introduced. Preferably, the antibody further comprises a light chain having the amino acid sequence shown in SEQ ID NO: 1.

[0037] In another embodiment, the heavy chain of the antibody of the invention has the amino acid sequence as set forth in SEQ ID NO: 2, into which the mutations E233P / L234V / L235A / 236del / S239D / A330L / I332E / M252Y / S254T / T256E have been introduced. Preferably, the antibody further comprises a light chain having the amino acid sequence as set forth in SEQ ID NO: 1.

[0038] In another embodiment, the heavy chain of the antibody of the invention has the amino acid sequence as set forth in SEQ ID NO: 2, into which the mutations E233P / L234V / L235A / 236del / K326A / I332E / E333A / N434A have been introduced. Preferably, the antibody further comprises a light chain having the amino acid sequence as set forth in SEQ ID NO: 1.

[0039] In another embodiment, the heavy chain of the antibody of the invention has the amino acid sequence set forth in SEQ ID NO: 25. Preferably, the antibody further comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 24.

[0040] In another embodiment, the heavy chain of the antibody of the invention has the amino acid sequence set forth in SEQ ID NO: 26. Preferably, the antibody further comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 24.

[0041] In another embodiment, the heavy chain of the antibody of the invention has the amino acid sequence set forth in SEQ ID NO: 27. Preferably, the antibody further comprises a light chain having the amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 24.

[0042] In a preferred aspect of the invention, the antibody of the invention is a non-fucosylated antibody or an antibody with reduced fucosylation.

[0043] As used herein, the term "antibody with reduced fucosylation" refers to an antibody in which less than 90% of the antibody's N-glycans are fucosylated. Methods for measuring the percentage of fucosylation are known in the art. In one embodiment, less than 75%, or less than 50%, or less than 25% of the antibody's N-glycans are fucosylated. Most preferably, less than 10% of the antibody's N-glycans are fucosylated. In a particular embodiment, the N-glycans of the antibody of the present invention do not contain any fucose.

[0044] 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.

[0045] In another embodiment, the N-glycan at N297 of the antibody does not contain any fucose.

[0046] Non-fucosylated antibodies, sometimes called afucosylated antibodies, can be produced by various methods. For example, synergistic knockdown of genes for α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in CHO cells can be used to produce fully non-fucosylated, ADCC-enhanced monoclonal antibody variants (see, e.g., Imai-Nishiya et al. (2007) BMC Biotechnol. 7, 84). A method using zinc finger nucleases (ZFNs) to cleave the FUT8 gene in the region encoding the catalytic core of α1,6-fucosyltransferase, thereby destroying the corresponding enzyme function in CHO cells, can be used to produce monoclonal antibodies that are completely devoid of core fucose (see, e.g., Malphettes et al. (2010) Biotechnol. Bioeng. 106, 774-783).

[0047] Antibodies with reduced fucosylation can be generated by adding a decoy substrate, such as 2-deoxy-2-fluoro-2-fucose, to the culture medium to reduce the incorporation of fucose into IgG-Fc glycans (see, e.g., Dekker et al. (2016) Sci Rep 6:36964).

[0048] In another embodiment, the antibodies of the present invention have high sialic acid content. Increased sialylation can be achieved, for example, by co-transfection of cytidine monophosphate-sialic acid synthase (CMP-SAS), cytidine monophosphate-sialic acid transporter (CMP-SAT), and alpha 2,3-sialyltransferase (see, for example, Son et al. (2011) Glycobiology 21, 1019-1028).

[0049] Affinity for FcRn The affinity of the antibodies of the present invention for human FcRn at pH 6 is high. High affinity binding of antibodies to human FcRn at pH 6 typically has a K of less than 500 nM. D Preferably, the K value for high affinity binding at pH 6 is D For example, the K value characterizing affinity at pH 6 is less than 400 nM, or less than 300 nM, or less than 200 nM. D Values ​​may range from 5 to 500 nM, or 10 to 400 nM, or 25 to 300 nM, or 50 to 200 nM, or 100 to 175 nM.

[0050] In a preferred embodiment, the affinity of the antibodies of the invention for human FcRn at pH 6 is greater than the affinity of infliximab for human FcRn at pH 6.0.

[0051] The affinity of the antibody of the present invention for human FcRn is preferably measured by surface plasmon resonance (SPR) as described, for example, in Example 4 of the present application.

[0052] The antibodies of the present invention typically have low affinity for human FcRn at pH 7.4. Low affinity is defined as a K D Preferably, the low affinity for human FcRn at pH 7.4 is characterized by a K value of greater than 2 μM, or greater than 5 μM, or greater than 10 μM. D It is characterized by its value.

[0053] In certain embodiments, the low affinity at pH 7.4 isD The value is so low that it cannot be measured by SPR.

[0054] In a specific embodiment, (i) the K of binding of an antibody of the invention to human FcRn at pH 7.4 D (ii) K of binding to human FcRn at pH 6.0 D is at least 50. Preferably, the ratio is at least 100, or at least 150, or at least 200.

[0055] Functional properties of antibodies The antibodies of the invention are efficiently transported across polarized cell monolayers from the apical to the basolateral side. Typically, transport across polarized cell monolayers is greater than that of infliximab, which has an antibody content of 10 ... 2 Relative to the amount of infliximab transported across the polarized cell monolayer, the amount of antibody transported across the polarized cell monolayer is at least 110%, preferably at least 120%, more preferably at least 130%, or at least 140%, or at least 150% (taking the amount of infliximab transported as 100%).

[0056] Furthermore, antibodies are specifically transported across polarized cell monolayers from the apical to the basolateral side in the presence of excess competing immunoglobulins, referred to herein as specific transport.

[0057] The percentage of the total mass of immunoglobulin transported across the polarized cell monolayer is greater than the percentage of infliximab transported across the polarized cell monolayer from apical to basolateral in the presence of a 10-fold excess of a competing immunoglobulin. The percentage of the antibody of the invention transported across the polarized cell monolayer in the presence of a 10-fold excess of an unrelated immunoglobulin is at least 120%, or at least 130%, or at least 140%, or at least 150% (infliximab taken as 100%) relative to the percentage of infliximab transported across the polarized cell monolayer in the presence of a 10-fold excess of an unrelated immunoglobulin.

[0058] Preferably, the polarized cell monolayer is a monolayer of polarized T84 cells.The transcytotic transport assay mimicking process can be carried out as described in Example 5 of the present application.

[0059] The antibodies of the invention bind to CD16a(V), CD16a(F) and CD16b(NA2).

[0060] Antibodies of the invention typically have a K for CD16a(V) of less than 1 μM, preferably less than 500 nM, more preferably less than 100 nM. D Combine with.

[0061] Antibodies of the invention typically have a K for CD16a(F) of less than 10 μM, preferably less than 1 μM. D Combine with.

[0062] Antibodies of the invention typically have a K for CD16b(NA2) of less than 10 μM, preferably less than 1 μM. D Combine with.

[0063] The antibody of the present invention further comprises + CD206 + Macrophages can be induced, preferably at a level equivalent to, the same as, or greater than that of infliximab.

[0064] The antibody of the present invention can further suppress T cell proliferation, preferably at a level equivalent to, the same as, or greater than that of infliximab.

[0065] Pharmaceutical Compositions and Treatments Treatment of a disease includes treatment of a patient already diagnosed with any form of the disease at any clinical stage or symptom; delaying the onset, or progression, or exacerbation, or deterioration of symptoms or signs of the disease; and / or preventing and / or reducing the severity of the disease.

[0066] A "subject" or "patient" to whom an anti-TNFα antibody is administered can be a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.), or a primate (e.g., monkey or human). In certain embodiments, the human is a pediatric patient. In other embodiments, the human is an adult patient.

[0067] Described herein are compositions 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 usually supplied as part of a sterile pharmaceutical composition that includes a pharma- ceutically acceptable carrier. The composition can be in any suitable form (depending on the desired method of administration to a patient).

[0068] Anti-TNFα antibodies can be administered to patients by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intrathecal, topically or locally, e.g., transmucosally. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. In one embodiment, anti-TNFα antibodies are administered intravenously.

[0069] In a particularly preferred embodiment, the antibodies of the invention are administered orally. When administration is by the oral route, the antibody is preferably an IgG, most preferably an IgG1.

[0070] The anti-TNFα antibody may be present in the pharmaceutical composition at a concentration sufficient to allow for intravenous administration of 0.5 mg / kg to 20 mg / kg body weight. In some embodiments, concentrations of the antibody suitable for use in the compositions and methods described herein include, but are not limited to, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, or concentrations ranging between any of the aforementioned 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.

[0071] An effective dose of anti-TNFα antibody may range from about 0.001 to about 750 mg / kg per single (e.g., bolus), multiple, or continuous administration, or may range to achieve a serum concentration of 0.01 to 5000 μg / mL per single (e.g., bolus), multiple, or continuous administration, or may be any effective range or value therein, depending on the condition being treated, the route of administration, and the age, weight, and condition of the subject. In the case of oral administration, the serum concentration may be very low or below the limit of detection. In certain embodiments, each dose may range from about 0.5 mg / kg body weight to about 50 mg / kg body weight or from about 3 mg / kg body weight to about 30 mg / kg body weight. The antibody may be formulated as an aqueous solution.

[0072] In a particularly preferred embodiment, the antibody of the invention is administered orally. When administration is by the oral route, the antibody is preferably an IgG, most preferably an IgG1. When the antibody is administered orally, the daily dose of the antibody is usually in the range of about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.05 mg / kg body weight to about 50 mg / kg body weight, or about 0.1 mg / kg body weight to about 25 mg / kg body weight, or about 0.15 mg / kg body weight to about 10 mg / kg body weight, or about 0.16 mg / kg body weight to about 5 mg / kg body weight, or about 0.2 mg / kg body weight to about 2 mg / kg body weight, or about 0.2 mg / kg body weight to about 1 mg / kg body weight. Usually, advantageous dosages are those of 1 to 200 mg / day, preferably 5 to 100 mg / day or 10 to 50 mg / day.

[0073] The pharmaceutical composition can be conveniently provided in a unit dosage form containing a predetermined amount of anti-TNFα antibody per dose. Such a unit can 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 aforementioned values, for example, 10 mg to 1000 mg, 20 mg to 50 mg, or 30 mg to 300 mg. Pharmaceutically acceptable carriers can take a wide variety of forms, depending, for example, on the condition to be treated or the route of administration.

[0074] Determining an effective dosage of anti-TNFα antibody, the total number of doses administered, and the length of treatment period is well within the capabilities of one of ordinary skill in the art, and can be determined using standard dose escalation studies.

[0075] Therapeutic formulations of anti-TNFα antibodies suitable for the methods described herein can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibody having the desired purity with any pharma- ceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as "carriers") commonly used in the art, such as buffers, stabilizers, preservatives, isotonicity agents, non-ionic surfactants, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be non-toxic to the recipient at the dosages and concentrations used.

[0076] Buffers serve to maintain a pH in a range close to physiological conditions. They can be present in concentrations ranging from about 2 mM to about 50 mM. Suitable buffers include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), citrate-phosphate buffers, succinic acid buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumaric acid buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid- disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconic acid buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactic acid buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

[0077] 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, for example, about 150 mM.

[0078] Preservatives can be added to retard microbial growth and can be added in amounts ranging from 0.2% to 1% (w / v). Suitable preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and the like. Isotonicity agents, sometimes known as "stabilizers," can be added to ensure isotonicity of the liquid composition and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients that can serve a variety of functions, from bulking agents to additives that solubilize the therapeutic agent or help prevent denaturation or adhesion to the container walls. Typical stabilizers include polyhydric sugar alcohols (as listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols (cyclitols such as inositol), such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, etc.; polyethylene glycol; amino acid polymers; urea, glutathione, thiol, etc. The stabilizer may be a sulfur-containing reducing agent such as octoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; a protein such as a low molecular weight polypeptide (e.g., a peptide of 10 residues or less), human serum albumin, bovine serum albumin, gelatin, or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; a monosaccharide such as xylose, mannose, fructose, glucose; a disaccharide such as lactose, maltose, sucrose; a trisaccharide such as raffinose; and a polysaccharide such as dextran. The stabilizer may be present in the range of 0.1 to 10,000 parts by weight per part by weight of active protein.

[0079] A non-ionic surfactant or detergent (also known as a "wetting agent") can be added to aid in solubilizing the therapeutic agent, to protect the therapeutic protein from agitation-induced aggregation, and to allow the formulation to be exposed to stressed shear surfaces without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), pluronic acid polyols, polyoxyethylene sorbitan monoethers (Tween®-20, Tween®-80, etc.). The non-ionic surfactant can be present in a range of about 0.05 mg / ml to about 1.0 mg / ml, or in a range of about 0.07 mg / ml to about 0.2 mg / ml.

[0080] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), protease inhibitors, and cosolvents.

[0081] The formulations herein may also include a second therapeutic agent in addition to the anti-TNFα antibody. Examples of suitable second therapeutic agents are provided below.

[0082] The administration schedule can vary from once a month to daily, depending on a number of clinical factors, such as the type of disease, the severity of the disease, and the patient's sensitivity to the anti-TNFα antibody. In certain embodiments, the anti-TNFα antibody is administered once a day, twice a week, three times a week, every other day, every 5 days, once a week, every 10 days, every 2 weeks, every 3 weeks, every 4 weeks, or once a month, or within any range between any two of the above values, such as every 4 days to once a month, every 10 days to every 2 weeks, or 2-3 times a week.

[0083] The dosage of anti-TNFα antibody to be administered will vary depending on the particular antibody, the subject, as well as the nature and severity of the disease, the subject's health status, the therapy (e.g., whether a second therapeutic agent is used), and the selected route of administration, and appropriate dosages can be readily determined by one of skill in the art.

[0084] Those skilled in the art will understand that the optimal amount and interval of each dosage of anti-TNFα antibody will be determined by the nature and severity of the condition being treated, the form of administration, the route of administration, and the site of administration, as well as the age and condition of the particular subject being treated, and the doctor will ultimately determine the appropriate dosage to be used. This dosage can be repeated as many times as necessary. If side effects occur, the amount and / or frequency of the dosage can be changed or reduced according to normal clinical practice.

[0085] Disorders to be treated The present invention relates to a method of treating or preventing a human TNFα-related disease in a subject comprising administering to the subject an antibody as defined herein. The term "TNFα-related disorder" or "TNFα-related disease" refers to any disorder, onset, progression or persistence of a symptom or disease state that requires the involvement of TNFα. Exemplary TNFα-related disorders include, but are not limited to, chronic and / or autoimmune conditions of inflammation generally, immune-mediated inflammatory diseases generally, inflammatory CNS diseases, inflammatory diseases affecting the eyes, joints, skin, mucous membranes, central nervous system, gastrointestinal tract, urinary tract, or lungs, uveitis conditions generally, retinitis, HLA-B27+ uveitis, Behcet's disease, dry eye syndrome, glaucoma, Sjogren's syndrome, diabetes mellitus (including diabetic neuropathy), insulin resistance, arthritic conditions generally, rheumatoid arthritis, vascular endothelial cell carcinoma ... Osteoarthritis, reactive arthritis and Reiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiple sclerosis, Guillain-Barre 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, pustulosis, hyperostosis and osteitis), acne, Sweet's syndrome, pemphigus, Crohn's disease (including extraintestinal manifestations), ulcerative colitis, bronchial asthma, hypersensitivity pneumonitis, total bladder inflammation, psoriasis (including psoriatic arthritis), hidradenitis suppurativa, panniculitis, pyoderma gangrenosum ... total bladder inflammation, psoriasis (including psoriatic arthritis), hidradenitis suppurativa, panniculitis, pyoderma gangrenosum, total bladder inflammation, psoriasis (including psoriatic arthritis), hidradenitis suppurativa, panniculitis, total bladder inflammation, psoriasis (including psoriatic arthritis), hidradenitis suppurativa, panniculitis, total bladder inflammation, psoriasis (including psoriatic arthritis), hidradenitis suppurativa, panniculitis, total bladder inflammation, psoriasis (including psoriatic arthritis), Allergies, allergic rhinitis, allergic sinusitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, Wegener's granulomatosis, Kawasaki syndrome, giant cell arteritis, Churg-Strauss vasculitis, polyarteritis nodosa, burns, graft-versus-host disease, host-versus-graft reaction, rejection episodes after organ or bone marrow transplantation, generalized systemic and localized conditions of vasculitis, systemic and cutaneous lupus erythematosus, polymyositis and dermatomyositis, scleroderma, preeclampsia, acute and chronic pancreatitis, viral hepatitis, alcoholic hepatitis, post-operative inflammation such as after eye surgery (e.g. cataract (eye lens replacement) or glaucoma surgery), joint surgery (including arthroscopic surgery), surgery on joint-related structures (e.g. ligaments), oral and / or dental surgery, minimally invasive cardiovascular procedures (e.g. PTCA, atherectomy, stent placement), laparoscopic and / or endoscopic intraperitoneal and gynecological procedures, endoscopic urological procedures (e.g. prostate surgery, ureteroscopy, cystoscopy, interstitial cystitis), or peri- and post-operative inflammation (prophylaxis) in general,These include bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, pustular dermatitis, cerebral malaria, hemolytic uremic syndrome, allograft rejection, otitis media, snakebite, erythema nodosum, myelodysplastic syndrome, primary sclerosing cholangitis, seronegative spondyloarthropathy, autoimmune hemolytic anemia, orofacial granulomatosis, vegetative suppurative stomatitis, aphthous stomatitis, geographic tongue, migratory stomatitis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Bell's palsy, Creutzfeldt-Jakob disease, as well as neurodegenerative conditions in general.

[0086] Cancer-associated osteolysis, cancer-associated inflammation, cancer-associated pain, cancer-associated cachexia, bone metastases, acute and chronic types of pain whether due to central or peripheral actions of TNFα and whether they are classified as inflammatory, pain of nociceptive or neuropathic type, sciatica, low back pain, carpal tunnel syndrome, complex regional pain syndrome (CRPS), gout, post-herpetic neuralgia, fibromyalgia, regional pain states, chronic pain syndromes due to metastatic tumors, dysmenorrhea.

[0087] Particular disorders to be treated include arthritic conditions in general, rheumatoid arthritis, osteoarthritis, reactive arthritis, juvenile arthritis, psoriasis such as psoriatic arthritis, inflammatory bowel disease such as Crohn's disease, ulcerative colitis such as proctitis, sigmoiditis, proctosigmoiditis, left-sided colitis, extensive colitis and pancolitis, indeterminate colitis, microscopic colitis such as collagenous and lymphocytic colitis, colitis in connective tissue diseases, diversion colitis, colitis in diverticular disease, eosinophilic colitis, and pouchitis.

[0088] Most preferably, the antibody of the present invention is used to treat inflammatory bowel disease, particularly Crohn's disease, ulcerative colitis, or microscopic colitis. Crohn's disease can be ileal, colonic, ileocolonic, or isolated upper Crohn's disease (stomach, duodenum, and / or jejunum), including non-stenotic / non-permeable, stenotic, permeable, and perianal disease behaviors, and any combination of any of the above localizations and disease behaviors is possible. Ulcerative colitis can be ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolonic ulcerative colitis, and pouchitis.

[0089] Combination Therapy and Other Aspects Preferably, the patient treated with anti-TNFα antibody is also treated with another conventional drug.For example, inflammatory bowel disease patients, especially those with moderate to severe disease, are usually also treated with mesalazine or its derivatives or prodrugs, budesonide, or corticosteroids such as prednisolone (oral or intravenous), immunosuppressants such as azathioprine / 6-mercaptopurine (6-MP) or methotrexate, cyclosporine or tacrolimus.Other drugs that can be administered to patients at the same time include other anti-TNFα antibodies (e.g., infliximab, adalimumab, etanercept, certolizumab pegol, golimumab), integrin antagonists (e.g., natalizumab, vedolizumab), anti-IL-23 antibodies (e.g., MEDI2070), anti-β7 antibodies (e.g., etrolizumab), JAK inhibitors of JAK / STAT pathway (e.g., tofacitinib), etc. Further drugs that may be co-administered to the patient include immunosuppressants (e.g., azathioprine / 6-MP or methotrexate or oral cyclosporine) to maintain stable, longer-term remission. Yet another aspect of the present invention is the use of an anti-TNFα antibody as defined herein above to reduce inflammation.

[0090] Yet another aspect of the present invention is an anti-TNFα antibody as defined herein above for use in reducing inflammation in a patient suffering from an inflammatory condition.

[0091] A further aspect of the invention is a method for treating an inflammatory condition, comprising administering to a patient in need thereof an effective amount of an anti-TNFα antibody as defined herein above, the inflammatory condition being preferably one of the above mentioned conditions.

[0092] A further aspect of the invention is a method for preventing an inflammatory condition, comprising administering to a patient in need thereof an effective amount of an anti-TNFα antibody as defined herein above, the inflammatory condition being preferably one of the above mentioned conditions.

[0093] Yet another aspect of the invention is a method for improving the transcytosis of an antibody against TNFα, comprising introducing the substitutions E233P, L234V and L235A, deleting G236 and adding the following further substitutions (a) or (b) to obtain a modified antibody with improved transcytosis: (a) M252Y, S254T and T256E, (b) N434A, and, optionally, further introducing one or more other substitutions as described herein. The modified antibody is preferably an antibody as described herein above.

[0094] Yet another aspect of the invention is a method for increasing the plasma half-life of an antibody against TNFα, comprising introducing the substitutions E233P, L234V and L235A, deleting G236 and adding the following further substitutions (a) or (b) to obtain a modified antibody with increased plasma half-life: (a) M252Y, S254T and T256E, (b) N434A, and, optionally, further introducing one or more other substitutions as described herein. The modified antibody is preferably an antibody as described herein above. The plasma half-life may be extended by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to the plasma half-life of the unmodified antibody (i.e., the respective parent antibody lacking the described mutations).

[0095] Yet another aspect of the invention is a method for improving the resistance to proteolysis of an antibody against TNFα, comprising introducing the substitutions E233P, L234V and L235A, deleting G236 and adding the further substitutions (a) or (b) below, in order to obtain a modified antibody having improved resistance to proteolysis: (a) M252Y, S254T and T256E, (b) N434A, and, optionally, further introducing one or more other substitutions as described herein. The modified antibody is preferably an antibody as described herein above. [Table 1]

[0096] Working Example Antibody variants Several variants of anti-TNFα antibody (hereafter referred to as “parent antibody” or “Ab-wt”) were generated by introducing substitutions in the Fc region of the antibody amino acid sequence. The light chain of Ab-wt has the amino acid sequence shown in SEQ ID NO:1, and the heavy chain of Ab-wt has the amino acid sequence shown in SEQ ID NO:2. Mutations were introduced by established methods of site-directed mutagenesis. Briefly, mutations were introduced by PCR. A forward primer was designed to contain the desired mutation, and at the same time, a reverse primer was designed such that the 5′ ends of the two primers were annealed back-to-back (but not overlapping) ( FIG. 10 ). 25 cycles of PCR were performed (98° C. for 10 s, 64° C. for 30 s, 72° C. for 3 min). Before the PCR products were subjected to agarose gel, the non-mutated PCR template was removed from the pool of PCR products using the restriction enzyme DpnI. 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 colonies were picked and plasmid DNA was isolated and sequenced to confirm that the mutation had been incorporated. [Table 2]

[0097] Example 1. Affinity for TNFα method: Affinity for TNFα was measured by Biacore. CM5 chips were prepared using standard amine immobilization Biacore procedures. Upon insertion of the CM5 chip, the system was primed and then normalized with BIA normalization solution (Biacore Preventative Maintenance Kit 2). The chip was added to the system with phosphate-buffered saline Tween-20 (PBS-T) running buffer; prior to immobilization, the chip surface was primed with three injections of 50 mM NaOH. Protein A was immobilized on the chip surface. For this, the protein was diluted to 5 μg / mL in 10 mM acetate buffer at pH 4.5 and injected to generate a binding response of approximately 1000 RU in all four flow cells. Three 15-second washes with 50 mM NaOH were performed to remove non-covalently bound material from all chip flow cells. On the Protein A chip, the antibody was captured in flow cells 2 and 4, while flow cells 1 and 3 were used for baseline subtraction. The test antibody was diluted to 10 nM in PBS-T and 2.5–7.5 uL were injected to obtain 120 RU of capture antibody. The analyte TNFα was made up at 500 μg / mL in water and further diluted in running buffer PBS-T according to the supplier's instructions. Single cycle kinetics were used to estimate steady-state affinity. For each single cycle analysis cycle, a titration of five analyte concentrations was injected over the ligand, followed by measurement of complex dissociation. The surface was regenerated using glycine pH 1.7. A double reference method was employed, where data from the ligand-bound capture surfaces (fc2 and 4) were subtracted from the reference surfaces with no ligand captured (fc1 and 3, respectively). Blank injections of buffer were performed every 3–4 cycles and then subtracted from the analyte injection cycles to correct for small changes in the ligand capture surface. Replicate injections of analyte at the beginning and end of each analytical run were used to investigate sample degradation, or changes in instrument performance. All analyses were performed at 25° C. and sample racks were incubated at 10° C. throughout the experiment. Each experiment was performed at least in triplicate. A one-to-one binding model was used to fit the resulting kinetic data.

[0098] result: All antibodies showed similar binding kinetics to TNFα and none of the introduced modifications resulted in major changes in the antigen-binding region. [Table 3]

[0099] Example 2. Efficacy method: L929 cells were incubated with 0.25 ng / mL TNFα and 1 μg / well actinomycin D in the presence of serial dilutions of anti-TNFα antibody variants. After 20 h of incubation at 37°C / 5% CO2, the proliferative response was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling reagent (phenazine ethosulfate, PES). MTS was converted to a formazan product by dehydrogenases present in metabolically active cells. The amount of formazan product, measured by absorbance at 492 nm, was directly proportional to the number of viable cells in the culture.

[0100] result: The results are shown in Figure 1. Introduction of mutations into the Fc region of the anti-TNFα antibody did not affect potency.

[0101] Example 3. Affinity for Fcγ receptors (CD16a, CD16b) method: Affinity for FcγR was measured by Biacore. CM5 chips were prepared using standard amine immobilization Biacore procedures. Upon insertion of the CM5 chip, the system was primed and then normalized with BIA normalization solution (Biacore Preventative Maintenance Kit 2). The chip was added to the system with PBS-T running buffer; prior to immobilization, the chip surface was primed with three injections of 50 mM NaOH. FcγR was immobilized on the chip surface using the His-tag capture system. Anti-His-tag chips were prepared according to the Biacore kit instructions, and approximately 12000 RU of antibody was deposited on all four flow cells. Three 30-second washes with 10 mM glycine pH 1.5 were performed to remove non-covalently bound material from all chip flow cells. Fcγ receptors were diluted in PBS-T in the range of 0.5-2 μg / mL and 2.5-5.0 μL was injected onto the chip, generating capture levels of 60-200 RU. Antibodies were diluted in PBS-T prior to analysis. Single cycle kinetics were used to estimate steady state affinity. For each single cycle analysis cycle, a titration of five antibody concentrations was injected over the FcγR ligand, followed by measurement of complex dissociation. The recommended solution, 10 mM glycine pH 1.5, was used to regenerate the surface for the anti-His capture surface. A double reference method was employed, where data from ligand-bound capture surfaces (fc2 and 4) were subtracted from reference surfaces with no ligand captured (fc1 and 3, respectively). A buffer blank injection was performed for every antibody titration cycle, then subtracted from the analyte injection cycle to correct for small changes in the ligand capture surface. All analyses were performed at 25° C., and the sample rack was incubated at 10° C. during the experiment. Each experiment was performed at least in triplicate.

[0102] result: The introduction of mutations did not affect the affinity for CD16a(V), CD16a(F) and CD16b. However, some antibody variants showed increased binding to CD16a. In particular, Ab-A-DLE-PVAΔG improved binding to the low affinity CD16a receptor and CD16b. [Table 4]

[0103] Example 4. Affinity for FcRn method: SPR was performed on a Biacore3000 instrument using a CM5 sensor chip coupled with anti-TNFα IgG1 antibody (approximately 500 resonance units (RU)) using amine coupling chemistry as described by the manufacturer. Coupling was performed by injecting 2.0ug / mL of each protein in 10mM sodium acetate, pH 4.5 using an amine coupling kit (GE Healthcare). 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) were used as running and dilution buffers. Binding kinetics were determined by injecting titrating amounts (1000-31.2nM) of monomeric His-tagged human FcRn (hFCRn) over the immobilized antibody at pH 7.4 or pH 6.0. All SPR experiments were performed at 25° C. with a flow rate of 40 ul / min. Binding data were zero adjusted and reference cell values ​​were subtracted. Binding kinetics were determined using the Langmuir 1:1 ligand binding model provided by the BIAevaluation software (version 4.1).

[0104] result: The results showed that the wild-type antibody Ab-wt bound to hFcRn in a strictly pH-dependent manner. All engineered antibody variants had higher affinity for FcRn at pH 6.0, but retained their pH-dependence and did not bind to the receptor at pH 7.4. All antibody variants showed improved binding to FcRn compared to infliximab, which contains the wild-type IgG1 Fc region. [Table 5]

[0105] Example 5. Transcytosis method: A transwell filter (1.12 cm) with a 0.4 μm-sized collagen-coated polytetrafluoroethylene (PTFE) membrane was 2 ) were incubated overnight in complete growth medium followed by 1.0x10 cells per well. 6 T84 cells were seeded. Transepithelial electrical resistance (TEER) was monitored daily using a Millicell-ERS-2 volt-ohm meter. After the cultures were grown for 4-5 days, they reached approximately 1000-1300 Ωxcm 2 Confluence was reached with a TEER value of 0.01%. Prior to the experiment, the monolayers were starved for 1 h in Hank's Balanced Salt Solution (HBSS). Then, 400 nM of antibody variants or IFX alone or together with 4000 nM of human myeloma IgG with irrelevant specificity were added to the apical transwell chamber. Samples were collected from the basolateral reservoir at 0 and 4 h after addition. Antibody concentrations in the basolateral reservoir were measured by ELISA. Briefly, 96-well Maxisorp plates were coated overnight with recombinant TNFα or goat-derived anti-human Fc specific antibodies (both diluted in PBS to 1 μg / ml). Plates were then blocked with 4% skim milk in PBS for 2 h at room temperature, followed by 4 washes with 0.05% Tween 20 in PBS. Samples collected during the transcytosis experiments were added to the wells and incubated for 2 h at room temperature, followed by washing as above. The captured antibody variants, IFX or total IgG were detected using an alkaline phosphatase (ALP)-conjugated goat anti-human Fc specific antibody. Binding was visualized by addition of 100 μl of ALP-substrate and the absorption spectrum was recorded at 405 nm. The amount of transferred antibody variants, IFX and total IgG was calculated from the calibration curves of each individual antibody variant.

[0106] Transcytosis of antibody variants across polarized human epithelial cells result: The genetically engineered anti-TNFα antibody variants were tested for transcytosis across cell monolayers and compared to another human IgG1 anti-TNFα antibody, the wt antibody or IFX. The results are shown in Figure 2. The wt anti-TNFα antibody was transported from the apical to the basolateral reservoir. Among the panel of genetically engineered variant antibodies to improve binding to FcRn, somewhat more Ab-YTE-DLE-PVAΔG was shown to be released at the basolateral side compared to Ab-wt. Compared to IFX, another IgG1 antibody with a wt Fc region, Ab-A-DLE-PVAΔG, was transported approximately 1.8-fold more efficiently.

[0107] Transcytosis of antibody variants across polarized human epithelial cells in the presence of competing IgG result: When anti-TNFα antibody variants were incubated with a 10-fold excess of human myeloma IgG 4 hours after addition, the total amount of immunoglobulin transported across polarized T84 cell monolayers from the apical to the basolateral reservoir was comparable for all antibodies. However, the increased affinity for FcRn at pH 6.0 resulted in a significantly higher percentage of specific anti-TNFα transport across the cell monolayer, even in the presence of excess competing human IgG with irrelevant specificity. The results are shown in Figure 3.

[0108] Example 6. ADCC method: The ADCC Reporter Bioassay Core Kit from Promega was used. Briefly, mTNFα CHO-K1 target cells were cultured at 1x10 5 100 μL per well was seeded into white (clear bottom) tissue culture dishes at 100 / mL. Plates were incubated overnight at 37°C / 5% CO2. On day 2, 95 μL of assay medium was removed and 25 μL of 3x10 61 / mL of genetically modified Jurkat effector cells were substituted. Plates were then incubated at 37°C / 5% CO2 for 6 hours. Towards the end of the incubation, BioGlo™ Reagent was made. Plates were equilibrated at room temperature for 10-20 minutes before adding 75 μL of BioGlo™ Reagent per well. Luminescence was measured in the dark after 5-10 minutes of incubation. The data was fitted using a 4-PL model.

[0109] result: The results (see FIG. 4) showed that all anti-TNFα antibodies induced ADCC, but with different intensities. Compared to the wild-type antibody Ab-wt, Ab-A-AEA-PVAΔG showed similar ADCC activity, while the other antibody variants showed increased ADCC. In particular, Ab-A-DLE-PVAΔG had significantly improved ADCC.

[0110] Example 7. Induction of regulatory macrophages method: Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors. Cells were isolated by Ficoll density gradient centrifugation. Cells from two individual donors were mixed in equal numbers and 2x10 cells of the mixture were cultured. 5Cells were seeded in 96-well plates in a total volume of 100 μL / well. Cells were incubated for 48 hours at 37°C / 5% CO2. After 48 hours, anti-TNFα antibody variants or IFX were added to reach a final concentration of 10 μg / mL. Each compound was added in 5 or 6 replicates. The final volume was 150 μL / well. Human serum IgG1 (Sigma #I5154) was used as a control. After compound addition, the mixed lymphocyte reactions (MLRs) were cultured for another 4 days at 37°C / 5% CO2. Afterwards, plates were washed with PBS / 5mM EDTA (PBS / EDTA) and incubated with 50 μL / well PBS / EDTA for 20 minutes at room temperature. Plates were centrifuged and splashed. Antibodies were diluted in PBS / EDTA (anti-CD14-PE, anti-CD206-APC, both diluted 1:10). Cells were resuspended in 50 μL of antibody solution and incubated for 20 min at room temperature. Afterwards, cells were washed with PBS / EDTA and resuspended in 50 μL of PBS / EDTA. Stained samples were analyzed on a FACS Fortessa using FACSDiva software. Analysis was performed using FlowJo software.

[0111] result: Regulatory macrophage induction was analyzed in four independent MLRs and was successful in all experiments (IFX vs. IgG control). Results are shown in Figure 5. The level of induction by IFX may vary between experiments due to the fact that each experiment was performed using a different donor with inter-individual variability. All tested anti-TNFα antibody variants inhibited CD14 + CD206 + Regulatory macrophages were induced with little variation between compounds. Ab-A-DLE-PVAΔG induced more regulatory macrophages than IFX.

[0112] Example 8. Inhibition of T cell proliferation method: PBMCs were isolated from buffy coats of healthy donors. Cells were isolated by Ficoll density gradient centrifugation. Cells from two individual donors were mixed in equal numbers and 2x10 of the mixture were cultured. 5Cells were seeded in 96-well plates in a total volume of 100 μL / well. Cells were incubated for 48 hours at 37°C / 5% CO2. After 48 hours, anti-TNFα antibody variants or IFX were added to reach a final concentration of 10 μg / mL. Each compound was added in 5 or 6 replicates. The final volume was 150 μL / well. Human serum IgG1 (Sigma #I5154) was used as a control. After compound addition, the mixed lymphocyte reaction (MLR) was cultured for an additional 2 days at 37°C / 5% CO2. Afterwards, tritiated thymidine ( 3 H thymidine (0.5 microcuries / well) was added to the cultures. Cultures were further incubated at 37°C / 5% CO2 for 18 hours. Samples were harvested using a Microbeta Filtermat96 cell harvester and analyzed using a Microbeta MicroplateCounter equipped with a single detector. Samples were counted for 10 seconds / well and converted to counts per minute (cpm).

[0113] result: Inhibition of T cell proliferation was measured in three independent MLRs and success was defined if IFX as a positive control induced suppression. The level of suppression by IFX in individual experiments may differ, possibly due to variability in regulatory macrophage induction. In each experiment, the potential of anti-TNFα antibody variants to suppress T cell proliferation was calculated relative to the positive control IFX. The antibody Ab-A-DLE-PVAΔG showed significantly enhanced suppression compared to IFX, whereas the suppression by Ab-YTE-DLE-PVAΔG was comparable to IFX (see Figure 6).

[0114] Example 9. Protease stability method: The analysis was carried out under reducing and non-reducing conditions. The reaction was quenched with the corresponding sample buffer from the PerkinElmer Protein Express Reagent Kit, with and without the reducing agent DTT (i.e., used as a stop reagent).

[0115] To be able to distinguish between the variants, the amount of protease per IgG was chosen such that a degradation-time profile was obtained over a period of 30 h. The analysis was carried out using a microchip-based electrophoresis system.

[0116] IdeS digestion To make working solution (ws), one aliquot of IdeS was reconstituted in 100 μL of milli-Q water. IdeS ws and sample were mixed in a 1:1 (v / v) ratio and homogenized thoroughly. The solution was incubated at 37°C and samples were removed after 5, 10, 30, and 60 min and quenched with one of the stop reagents. Protease / IgG molar ratio: 4:1.

[0117] GluC digestion To make working solution (ws), GluC stock was diluted with 2X GluC reaction buffer to a concentration of 50 μg / mL. GluC and samples were mixed in a 1:1 (v / v) ratio and thoroughly homogenized. The solution was incubated at 37°C and samples were removed after 2, 6, 24, and 30 hours and quenched with one of the stop reagents. Molar ratio of IgG / protease: 4:1.

[0118] MMP-3 digestion Chymotrypsin stock was diluted to 50 μg / mL in assay buffer MMP-3. Chymotrypsin diluted 1:1 (v / v) was added to 0.186 mg / mL MMP-3 and incubated at 37° C. for 30 min. Activation was stopped with PMSF at a final concentration of 2 mM. To make the working solution (ws), activated MMP-3 was diluted to 3.72 μg / mL in assay buffer.

[0119] MMP-3 ws and samples were mixed in a 1:1 (v / v) ratio and homogenized thoroughly. The solution was incubated at 37°C, and samples were removed after 2, 6, 24, and 30 hours and quenched with one of the stop reagents. Molar ratio of IgG / protease: 98:1.

[0120] result: The antibody variants tested showed excellent resistance to proteolysis by MMP-3 and IdeS, and good resistance to degradation by GluC (see Figures 7-9).

Claims

1. A humanized antibody comprising a TNFα binding domain and an FcRn binding site, the amino acid sequence of the antibody being: (i) contains amino acids 233P, 234V, 235A and 434A (EU numbering) and contains a deletion at amino acid position 236 (EU numbering); or (ii) comprising the amino acids 233P, 234V, 235A, 252Y, 254T and 256E (EU numbering), characterized in that it comprises a deletion at amino acid position 236 (EU numbering); The antibody, (iii) a V L domain comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:14, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:15, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:16; (iv) a VH domain comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:17, a CDR2 region having the amino acid sequence set forth in SEQ ID NO:18, and a CDR3 region having the amino acid sequence set forth in SEQ ID NO:19; and (v) an Fc region derived from human IgG1 An antibody comprising:

2. 2. The antibody of claim 1, wherein the amino acid sequence of the antibody further comprises amino acids 239D, 330L and 332E (EU numbering).

3. 2. The antibody of claim 1, wherein the amino acid sequence of the antibody further comprises amino acids 326A, 332E and 333A (EU numbering).

4. 2. The antibody of claim 1, wherein the amino acid sequence of the antibody comprises amino acids 233P, 234V, 235A, 239D, 330L, 332E and 434A (EU numbering) and contains a deletion at amino acid position 236 (EU numbering).

5. 2. The antibody of claim 1, wherein the amino acid sequence of the antibody comprises amino acids 233P, 234V, 235A, 239D, 330L, 332E, 252Y, 254T and 256E (EU numbering) and contains a deletion at amino acid position 236 (EU numbering).

6. 2. The antibody of claim 1, wherein the amino acid sequence of the antibody comprises amino acids 233P, 234V, 235A, 326A, 332E, 333A and 434A (EU numbering) and contains a deletion at amino acid position 236 (EU numbering).

7. Dissociation constant K<300 nM at pH 6 Ｄ and a dissociation constant K of greater than 10 μM at pH 7.

4. Ｄ The antibody according to any one of claims 1 to 6, which has no or low affinity for human FcRn, characterized by:

8. K<100 pM for human TNFα Ｄ The antibody according to any one of claims 1 to 7, which binds to

9. The antibody of any one of claims 1 to 8, which is transported across a polarized cell monolayer from the apical to the basolateral side in greater amounts than a control antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO:1 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:

2.

10. The antibody according to any one of claims 1 to 9, which is more resistant to proteolysis by MMP-3 and IdeS than infliximab.

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

12. An antibody according to any one of claims 1 to 10 for use in the treatment of an inflammatory condition.

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

14. The antibody for use according to claim 12, wherein the treatment comprises orally administering an effective amount of the antibody.

15. The antibody for use according to claim 12, wherein the antibody is applied topically.

16. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 10.

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