IgG1 ANTIBODY, PHARMACEUTICAL COMPOSITION AND USES OF THE REFERRED ANTIBODY

Abstract

The present invention relates to antibodies that bind to TNF-α and exhibit binding to modified PCR. The antibodies of the invention have good effector functions and / or pharmacokinetic properties.

Description

1 / 79 IgGi Antibody, Pharmaceutical Composition and Uses of Said Antibody FIELD OF THE INVENTION

[001] The present invention relates to modified antibodies with altered effector functions and / or altered pharmacokinetic properties. Antibodies are useful in the therapeutic treatment of various disorders, particularly inflammatory conditions. BACKGROUND

[002] Monoclonal antibodies have gained increasing importance as therapeutic reagents in clinical medicine over the past 20 years. For many years, efforts to improve antibodies have focused on reducing their potential immunogenicity, leading to humanized or even fully human antibodies. Another approach aims to optimize antibodies by improving their effector functions. While direct effects are mediated by the antibody's antigen-binding variable region, indirect effects are mediated by the constant Fc region. Efforts to improve effector functions focus primarily on modulating the Fc region. In addition, it is desirable to improve the serum half-life of therapeutic antibodies, which can reduce the amount of antibodies needed and increase their convenience for patients by extending treatment intervals. Petition 870250092396, dated 09 / 10 / 2025, page 13 / 102 2 / 79

[003] For therapeutic applications, immunoglobulin G (IgG) has been the preferred class of choice for several reasons; IgGs are easy to purify, are relatively stable in storage, can be administered intravenously, have a prolonged biological half-life in vivo, and are capable of engaging in a range of biological effector functions, such as activation of complement-dependent cytotoxicity (CDC) and recruitment of effector cells through various receptor-Fc interactions (antibody-dependent cellular cytotoxicity; ADCC). Of the five classes of immunoglobulins, 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. A solved FcRn-Fc cocrystal structure showed that the interaction with Fc occurs in the IgG-CH2CH3 hinge region.This interaction occurs in a strictly pH-dependent manner at an acidic pH of 6.0-6.5 in endosomes. Bound IgG molecules are recycled back to the cell surface, where they are released into the circulation at a physiological pH of 7.4, while uncomplexed IgG molecules are destined for lysosomal degradation. This recycling is the mechanism for the prolonged half-life of IgG; modulation of the FcRnIgG interaction will therefore allow specific control of the half-life. Petition 870250092396, dated 09 / 10 / 2025, page 14 / 102 3 / 79 serum lives of immunoglobulin gamma and Fc fusion proteins.

[004] Depending on the application, it may be desirable to increase or decrease the serum residence time of IgG. For therapeutic applications, a longer half-life is desirable because it requires smaller doses and fewer injections. Several approaches to increase half-life have been investigated, including the use of polyethylene glycol (PEG), generation of albumin or Fc fusion proteins, and strengthening the FcRn-IgG interaction. PEGylated pharmaceuticals have been in the clinic since 1990, and PEGylation is an established technology for extending drug residence time in the blood. Since human serum albumin (HSA) is also recycled by FcRn through a pH-dependent interaction, various albumin fusion proteins to improve stability and half-life have also been produced. Furthermore, antibody fragments fused with albumin or albumin-binding domains have demonstrated prolonged serum residence time in preclinical studies. Generation of Fc fusion proteins is another strategy that endows proteins or peptides with properties similar to an intact antibody.

[005] Modifications of the Fc region that have been investigated are summarized in Saxena (2016) Frontiers in Immunology, vol. 7, article 580. Petition 870250092396, dated 09 / 10 / 2025, page 15 / 102 4 / 79

[006] There is a continuing need for antibodies with improved effector and / or pharmacokinetic functions. SUMMARY OF THE INVENTION

[007] The inventors of this application discovered that certain specific mutations in the Fc region of an antibody dramatically increase the antibody's affinity for FcRn at pH 6, while the affinity at pH 7.4 remains low. Antibodies with the mutations are expected to have improved pharmacokinetic properties. Furthermore, the antibodies exhibit superior effector functions compared to known antibodies, such as infliximab (IFX).

[008] The invention therefore relates to the object defined in the following items [1] to

[88] :

[009] [1] An antibody comprising a TNFα binding domain and an FcRn binding site, having a high affinity for human FcRn at pH 6, said high affinity being characterized by a dissociation equilibrium constant (Kd) less than 100 nM and having a low affinity for human FcRn at pH 7.4, said low affinity being characterized by a Kd greater than 1 μM, wherein the amino acid sequence of the antibody comprises the amino acid 434W.

[0010] [2] The antibody from item [1], wherein the antibody amino acid sequence further comprises amino acid 428E and / or amino acid 311R. Petition 870250092396, dated 09 / 10 / 2025, p. 16 / 102 5 / 79

[0011] [3] The antibody from item [1] or [2], wherein the antibody amino acid sequence comprises amino acids 311R, 428E and 434W.

[0012] [4] The antibody according to any of the preceding items, wherein the antibody is obtained by replacing asparagine with tryptophan at position 434 (N434W), optionally replacing glutamic acid with methionine at position 428 (M428E) and optionally replacing glutamine with arginine at position 311 (Q311R).

[0013] [5] The antibody according to any of the preceding items, wherein the antibody is obtained by replacing methionine with glutamic acid at position 428 (M428E).

[0014] [6] The antibody according to any of the previous items, wherein the antibody is obtained by replacing glutamine with arginine at position 311 (Q311R).

[0015] [7] The antibody according to any of the preceding items, comprising a heavy chain comprising the amino acid sequence, as shown in SEQ ID NO: 13.

[0016] [8] The antibody in accordance with any of the previous items, having an affinity for human FcRn at pH 6 that is greater than that of infliximab.

[0017] [9] The antibody in accordance with any of the above items, in which the aforementioned high affinity to FcRn Petition 870250092396, dated 09 / 10 / 2025, p. 17 / 102 Human 6 / 79 at pH 6 is characterized by a dissociation constant Kd less than 50 nM.

[0018]

[10] The antibody according to any of the previous items, in which the aforementioned high affinity to human FcRn at pH 6 is characterized by a dissociation constant Kd less than 25 nM.

[0019]

[11] The antibody according to any of the previous items, in which the aforementioned high affinity to human FcRn at pH 6 is characterized by a dissociation constant Kd less than 10 nM.

[0020]

[12] The antibody according to any of the preceding items, having an affinity for human FcRn at pH 6, characterized by a dissociation constant Kd in the range of 1 nM to 500 nM, or 2 nM to 100 nM, or 3 nM to 50 nM, or 4 nM to 25 nM, or 5 nM to 10 nM.

[0021]

[13] The antibody according to any of the preceding items, wherein the said high affinity, or the said Kd which characterizes the said high affinity, is determined by surface plasmon resonance (SPR).

[0022]

[14] The antibody in accordance with any of the preceding items, in which the aforementioned low affinity to human FcRn at pH 7.4 is characterized by a Kd greater than 10 pM. Petition 870250092396, dated 09 / 10 / 2025, p. 18 / 102 7 / 79

[0023]

[15] The antibody according to any of the previous items, wherein the said Kd which characterizes the affinity with human FcRn at pH 7.4 is determined by SPR.

[0024]

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

[15] , wherein the affinity with human FcRn at pH 7.4 is so low that a KD value cannot be determined by SPR.

[0025]

[17] The antibody according to any of the above items, which binds to human TNFα with Kd less than 200 pM.

[0026]

[18] The antibody according to any of the above items, which binds to human TNFα with Kd less than 100 pM.

[0027]

[19] The antibody according to any of the above items, which binds to human TNFq with Kd less than 50 pM.

[0028]

[20] The antibody according to any of the above items, which binds to human TNF® with a KD less than 25 pM.

[0029]

[21] The antibody according to any of the above items, which binds to human TNFq with Kd less than 10 pM.

[0030]

[22] The antibody according to any of the above items, which is transported through a Petition 870250092396, dated 09 / 10 / 2025, p. 19 / 102 8 / 79 Monolayer of cells polarized from the apical side to the basolateral side.

[0031]

[23] The antibody according to any of the preceding items, which is transported through a monolayer of polarized cells from the apical side to the basolateral side in greater quantity than a control antibody comprising a light chain with the amino acid sequence as shown in SEQ ID NO: 1 and a heavy chain with the amino acid sequence as shown in SEQ ID NO: 2.

[0032]

[24] The antibody according to any of the above items, which is transported through a monolayer of polarized cells from the apical side to the basolateral side in greater quantity than infliximab.

[0033]

[25] The antibody according to item

[24] , in which the amount of antibody transported across the polarized cell monolayer is more than twice the amount of infliximab transported across the polarized cell monolayer.

[0034]

[26] The antibody according to any of items

[23] to

[25] , wherein the said quantity refers to the mass of antibody transported across the monolayer of polarized cells within four hours.

[0035]

[27] The antibody according to any of items

[22] to

[26] , where the amount of antibody Petition 870250092396, dated 09 / 10 / 2025, p. 20 / 102 9 / 79 transported across the polarized cell monolayer is more than twice the amount of a parental immunoglobulin transported across the polarized cell monolayer, wherein the said parental immunoglobulin differs from the said antibody only in that its Fc region has only wild-type amino acids.

[0036]

[28] The antibody of any of the preceding items, wherein a greater percentage of the antibody than that of infliximab is transported across a monolayer of polarized cells from the apical to the basolateral side in the presence of a tenfold excess of competing immunoglobulins, wherein the percentage refers to the total mass of immunoglobulins transported across the monolayer of polarized cells.

[0037]

[29] The antibody of item

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

[0038]

[30] The antibody of item

[28] or

[29] , in which the percentage of antibody transported across the polarized cell monolayer is more than three times the Petition 870250092396, dated 09 / 10 / 2025, page 21 / 102 10 / 79 percent of infliximab transported across the polarized cell monolayer.

[0039]

[31] The antibody according to any of items

[22] to

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

[0040]

[32] The antibody according to any of the above items, which binds to CD64 with Kd less than 100 nM, preferably less than 10 nM.

[0041]

[33] The antibody according to any of the above items, which binds to CD32a (H) with KD less than 10 μM.

[0042]

[34] The antibody of any of the above items, which binds to CD32a (R) with a Kd less than 10 μM.

[0043]

[35] The antibody according to any of the above items, which binds to CD32b with a Kd less than 10 μM.

[0044]

[36] The antibody according to any of the above items, which binds to CD16a (V) with a Kd less than 500 nM, preferably less than 100 nM.

[0045]

[37] The antibody according to any of the above items, which binds to CD16a (F) with a Kd less than 10 μM, preferably less than 1 μM. Petition 870250092396, dated 09 / 10 / 2025, p. 22 / 102 11 / 79

[0046]

[38] The antibody according to any of the above items, which binds to CD16b (NA2) with a Kd less than 10 μM, preferably less than 1 μM.

[0047]

[39] The antibody that matches any of the above items, which binds to human C1q more strongly than infliximab.

[0048]

[40] The antibody in accordance with any of the above items, with a higher complement-dependent cytotoxicity than infliximab, compared to infliximab, in terms of relative EC50 and / or relative maximum death.

[0049]

[41] The antibody that meets any of the above criteria, capable of inducing CD14+CD206+ macrophages at a level equal to or greater than infliximab.

[0050]

[42] The antibody in accordance with any of the preceding items, capable of suppressing T cell proliferation to an equal or greater degree than infliximab.

[0051]

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

[0052]

[44] The antibody according to any of the preceding items, comprising (i) a VL domain comprising a CDR1 region having the amino acid sequence as shown in SEQ ID NO: 3, a CDR2 region having the amino acid sequence as shown in SEQ ID NO: 4 and a Petition 870250092396, dated 09 / 10 / 2025, page 23 / 102 12 / 79 CDR3 region with the amino acid sequence as shown in SEQ ID NO: 5 and (ii) a VH domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 6, a CDR2 region with the amino acid sequence as shown in SEQ ID NO: 7 and a CDR3 region with the amino acid sequence as shown in SEQ ID NO: 8.

[0053]

[45] The antibody according to any of the preceding items, comprising a VH domain with the amino acid sequence as shown in SEQ ID NO: 9 and a VL domain with an amino acid sequence as shown in SEQ ID NO: 10.

[0054]

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

[0055]

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

[43] , wherein said antibody comprises (i) a VL domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 14, a CDR2 region having the amino acid sequence as shown in SEQ ID NO: 15, and a CDR3 region with the amino acid sequence as shown in SEQ ID NO: 16, and (ii) a VH domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 17, a CDR2 region Petition 870250092396, dated 09 / 10 / 2025, page 24 / 102 13 / 79 with the amino acid sequence as shown in SEQ ID NO: 18 and a CDR3 region with the amino acid sequence as shown in SEQ ID NO: 19.

[0056]

[48] The antibody according to item

[47] , comprising a VH domain with the amino acid sequence as shown in SEQ ID NO: 20 and a VL domain with an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22)

[0057]

[49] The antibody according to item

[47] or

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

[0058]

[50] The antibody in accordance with any of the above items, wherein said antibody binds specifically to human TNFα.

[0059]

[51] The antibody in accordance with any of the above items, in which the said antibody does not bind significantly to TNFp.

[0060]

[52] The antibody according to any of the above items, wherein said antibody (i) binds to human TNFα with a dissociation constant (KD) less than 125 pM; (ii) is reactive with TNFα from Macaca mulatta and with TNFα from Macaca fascicularis; Petition 870250092396, dated 09 / 10 / 2025, p. 25 / 102 14 / 79 (iii) has greater potency than infliximab, as determined by an L929 assay; and / or (iv) is capable of binding to human TNFαTrimer in a stoichiometry (antibody:TNFATrimer) of at least 2.

[0061]

[53] The antibody according to any of the above items, which binds to TNFα from Macaca mulatta with KD less than 1 nM.

[0062]

[54] The antibody according to any of the above items, which binds to TNFα from Macaca fascicularis with Kd less than 1 nM.

[0063]

[55] The antibody in accordance with any of the preceding items, wherein the antibody potency to inhibit TNFα-induced apoptosis relative to that of infliximab (relative potency), determined in an L929 assay, is greater than 3.5 and wherein said relative potency is the ratio of the IC50 value in ng / mL of infliximab in the L929 assay to the IC50 value in ng / mL of the antibody in the L929 assay.

[0064]

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

[0065]

[57] The antibody according to any of the above items, where the melting temperature of Petition 870250092396, dated 09 / 10 / 2025, p. 26 / 102 15 / 79 variable antibody domain in scFv format, as determined by differential scanning fluorimetry, is at least 68 °C.

[0066]

[58] The antibody of any of the above items, where the melting temperature, determined by differential scanning fluorimetry, is at least 70 °C.

[0067]

[59] The antibody according to any of the above items, in which the antibody is able to block the interaction between human TNFα and TNF receptor I (TNFRI).

[0068]

[60] The antibody that meets any of the above criteria, where the antibody is able to block the interaction between human TNFα and TNF receptor II (TNFRII).

[0069]

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

[0070]

[62] The antibody according to any of the previous items, which is capable of inhibiting LPS-induced interleukin-ΐβ secretion from CD14+ monocytes.

[0071]

[63] The antibody according to item

[62] , in which the IC50 value to inhibit LPS-induced interleukin1β secretion is less than 1 nM. Petition 870250092396, dated 09 / 10 / 2025, p. 27 / 102 16 / 79

[0072]

[64] The antibody according to item

[63] , wherein the said IC50 value for inhibiting LPS-induced interleukin-ΐβ secretion, on a molar basis, is lower than that of adalimumab.

[0073]

[65] The antibody that matches any of the previous items, which is capable of inhibiting LPS-induced TNFα secretion from CD14+ monocytes.

[0074]

[66] The antibody according to item

[65] , in which the IC50 value to inhibit LPS-induced TNF« secretion is less than 1 nM.

[0075]

[67] The antibody according to item

[66] , wherein the said IC50 value for inhibiting LPS-induced TNF® secretion, on a molar basis, is lower than that of adalimumab.

[0076]

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

[0077]

[69] A nucleic acid that encodes the antibody according to any of the above items.

[0078]

[70] A vector or plasmid comprising nucleic acid according to item

[69] .

[0079]

[71] A cell comprising the nucleic acid of item

[69] or the vector or plasmid of item

[70] .

[0080]

[72] A method for preparing the antibody according to any of items [1] to

[68] , comprising Petition 870250092396, dated 09 / 10 / 2025, page 28 / 102 17 / 79 cultivate the cell from item

[71] in a medium under conditions that allow the expression of the nucleic acid that encodes the antibody and recovering the antibody from the cells or from the medium.

[0081]

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

[68] for use in the treatment of an inflammatory condition or disorder related to TNFα.

[0082]

[74] The antibody for use in accordance with item

[73] , in which the aforementioned inflammatory disorder is selected from the list of diseases and disorders listed in the Disorders to be treated section below.

[0083]

[75] The antibody for use in accordance with item

[73] , in which the aforementioned inflammatory disorder is an inflammatory disorder of the gastrointestinal tract.

[0084]

[76] The antibody for use in accordance with item

[75] , in which the aforementioned inflammatory disorder of the gastrointestinal tract is an inflammatory bowel disease.

[0085]

[77] The antibody for use in accordance with item

[75] or

[76] , where the aforementioned inflammatory disorder of the gastrointestinal tract is Crohn's disease.

[0086]

[78] The antibody for use in accordance with item

[77] , in which the aforementioned Crohn's disease is selected from the group consisting of ileal, colonic, ileocolonic and / or isolated upper Crohn's disease (gastric, duodenal and / or jejunal chromatism) and including non-stressful / non-penetrating, restrictive disease behavior, Petition 870250092396, dated 09 / 10 / 2025, page 29 / 102 18 / 79 penetrating and perianal, allowing any combination of location and disease behavior of any of those mentioned above.

[0087]

[79] The antibody for use in accordance with item

[75] or

[76] , wherein the inflammatory disorder of the gastrointestinal tract referred to is ulcerative colitis.

[0088]

[80] The antibody for use in accordance with item

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

[0089]

[81] The antibody for use in accordance with item

[75] or

[76] , wherein the inflammatory disorder of the gastrointestinal tract referred to is microscopic colitis.

[0090]

[82] The antibody for use in accordance with item

[73] , wherein the inflammatory disorder referred to is arthritis.

[0091]

[83] The antibody for use in accordance with item

[73] or

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

[0092]

[84] The antibody for use in accordance with any of items

[73] to

[83] , wherein the said method comprises oral administration of the antibody to an individual.

[0093]

[85] The antibody for use in accordance with any of items

[73] to

[84] , wherein the said method comprises topical application of the antibody. Petition 870250092396, dated 09 / 10 / 2025, p. 30 / 102 19 / 79

[0094]

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

[68] .

[0095]

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

[0096]

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

[0097] Figure 1: Potency of anti-TNFα antibody variants to neutralize human TNFα in the L929 assay. Dose-response curves are shown for anti-TNFα antibody variants and reference infliximab.

[0098] Figure 2: Transport of anti-TNFα IgG variants across polarized T84 cells. The amounts of anti-TNFα antibody variants and Infliximab (IFX) from the apical to the basolateral reservoir are shown 4 hours after addition. Presented as ng / cm2. Error bars indicate SD of two to four individual monolayers.

[0099] Figure 3: Transport of anti-TNFα IgG variants across polarized T84 cells in the presence of excessive amounts of IgG myeloma. The following are shown Petition 870250092396, dated 09 / 10 / 2025, page 31 / 102 20 / 79 quantities of anti-TNFα Ab and IFX 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. Presented as ng / cm2. Error bars indicate SD of three to four individual monolayers.

[00100] Figure 4: ADCC activity. ADCC induction by anti-TNFa antibody variants, wild-type antibody, and IFX.

[00101] Figure 5: Binding to human C1q. Binding of IFX and anti-TNFa antibody variants to human C1q. Each concentration was tested in duplicate. Error bars indicate SD.

[00102] Figure 6: CDC activity. Percentage of cell death for the various anti-TNFa variants compared to IFX. Each sample point is the average of 6 independent replicates.

[00103] Figure 7: Induction of CD14+CD206+ macrophages by each compound in relation to IFX induction. Summary data from 4 independent experiments. Bars represent mean, error bars represent SEM.

[00104] Figure 8: Suppression of T cell proliferation by each compound relative to IFX. Summary data from 3 independent experiments. Bars represent mean, error bars represent SEM. Petition 870250092396, dated 09 / 10 / 2025, page 32 / 102 21 / 79

[00105] Figure 9: Schematic representation of site-directed mutagenesis.

[00106] Figure 10: A schematic illustration of the dominant N-glycan forms linked to N297 of anti-TNFa antibody variants. The two N-glycan profiles that dominated the panel of anti-TNFa antibody variants tested were 4GlcNac-1Fuc-3Man and 4GlcNac-1Fuc-3Man-1Gal, while for the IgG variants produced in the presence of 2FF the same bi-antenna structures occurred except that these did not have the fucose. DETAILED DESCRIPTION

[00107] The present invention relates to an antibody that is capable of binding to TNFα and comprises an FcRn binding site. According to this application, an antibody comprises an FcRn binding site if it is capable of binding to FcRn, preferably human FcRn, at pH 6. Binding to FcRn at pH 6 can be determined by SPR, for example, as described in Example 4 of this application. If the binding of an antibody to FcRn at pH 6 can be detected by SPR, that antibody will have an FcRn binding site. The antibody of the invention has high affinity for human FcRn at pH 6, characterized by a dissociation equilibrium constant (Kd) of less than 100 nM. The antibody also has low affinity for human FcRn at pH 7.4, characterized by a Kd greater than 1 μM. The sequence of Petition 870250092396, dated 09 / 10 / 2025, p. 33 / 102 22 / 79 amino acids of the antibody comprise the amino acid tryptophan at position 434 (EU numbering).

[00108] Throughout the present specification and claims, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain and residues 1-113 of the heavy chain) (Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)). The EU numbering system or EU index is generally used when referring to a residue in a constant region of the immunoglobulin heavy chain (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, Maryland (1991) expressly incorporated herein by reference). Unless otherwise indicated, references to residue numbers in the variable domain of antibodies mean residue numbering by the Kabat numbering system.Unless otherwise indicated, references to residue numbers in the antibody constant domain mean residue numbering using the EU numbering system (see, for example, WO 2006 / 073941). Antibody Petition 870250092396, dated 09 / 10 / 2025, page 34 / 102 23 / 79

[00109] In the context of the present application, the term antibody is used synonymously with immunoglobulin (Ig), which is defined as a protein belonging to the IgG, IgM, IgE, IgA, or IgD class (or any subclass) and includes all conventionally known antibodies and their functional fragments. In the context of the present invention, a functional fragment of an antibody / immunoglobulin is defined as an antigen-binding fragment or other derivative of a parental antibody that essentially retains one or more of the properties of that parental antibody. An antigen-binding fragment or antigen-binding domain of an antibody / immunoglobulin is defined as a fragment (e.g., a variable region of an IgG) that retains the antigen-binding region. An antigen-binding region of an antibody is typically found in one or more hypervariable regions of an antibody, i.e., in the CDR-1, -2, and / or -3 regions.The antibodies of the present invention can be part of bi- or multi-functional constructions.

[00110] Preferably, the antibody is a monoclonal antibody. The term monoclonal antibody, as used herein, is not limited to antibodies produced through hybridoma technology. The term monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Antibodies Petition 870250092396, dated 09 / 10 / 2025, p. 35 / 102 24 / 79 monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant display and phage technologies, or a combination thereof. (Harlow and Lane, Antibodies, A Laboratory Manual CSH Press 1988, Cold Spring Harbor NY).

[00111] In other embodiments, including embodiments related to the in vivo use of anti-TNFa antibodies in humans, chimeric, primatized, humanized, or human antibodies may be used. In a preferred embodiment, the antibody is a human antibody or a humanized antibody, more preferably a human monoclonal antibody or a humanized monoclonal antibody.

[00112] In another particular embodiment, the antibody of the invention is an immunoglobulin, preferably an immunoglobulin G (IgG). The IgG subclass of the invention is not limited and includes IgG1, IgG2, IgG3 and IgG4. Preferably, the IgG of the invention is of subclass 1, 2 or 4, that is, it is an IgG1, IgG2, or IgG4 molecule, respectively. More preferably, the IgG of the invention is of subclass 1, that is, it is an IgG1 molecule. TNFa linking domain

[00113] The TNFα-binding domain of the antibody of the invention is not particularly limited. It can be derived from any antibody capable of binding to TNFα. Petition 870250092396, dated 09 / 10 / 2025, p. 36 / 102 25 / 79

[00114] Preferably, the antibody of the invention specifically binds to TNFα. As used herein, an antibody specifically recognizes or binds specifically to human TNFα when the antibody is able to discriminate between human TNFα and one or more reference molecules. Preferably, the IC50 value for binding to each of the reference molecules is at least 1,000 times greater than the IC50 value for binding to TNFα. In its most general form (and when no defined reference is mentioned), specific binding refers to the ability of the antibody to discriminate between human TNFα and an unrelated biomolecule, as determined, for example, according to specificity assay methods known in the art. Such methods comprise, but are not limited to, Western blots and ELISA tests. For example, a standard ELISA test can be performed.Typically, the determination of binding specificity is performed using not a single reference biomolecule, but a set of about three to five unrelated biomolecules, such as milk powder, BSA, transferrin, or the like. In one embodiment, specific binding refers to the antibody's ability to discriminate between human TNFα and human TNFP.

[00115] The antibody of the invention comprises a VL domain and a VH domain. The VL domain comprises a CDR1 region. Petition 870250092396, dated 09 / 10 / 2025, page 37 / 102 26 / 79 (CDRL1), a CDR2 region (CDRL2), a CDR3 region (CDRL3), and Framework regions. The Vh domain comprises a CDR1 region (CDRH1), a CDR2 region (CDRH2), a CDR3 region (CDRH3), and Framework regions.

[00116] The term CDR refers to one of the six hypervariable regions within the variable domains of an antibody that contribute primarily to antigen binding. One of the most widely used definitions for 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 definition of CDRs applies only to CDR1, CDR2, and CDR3 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), as well as CDR2 and CDR3 of the heavy chain variable domain (CDR H2, CDR H3 or H2, H3). CDR1 of the heavy chain variable domain (CDR H1 or H1), however, as used herein, is defined by the following residues (Kabat numbering): It begins at position 26 and ends before position 36.

[00117] In one embodiment of the present invention, the antibody of the invention is an anti-TNFa antibody as disclosed in any of the PCT applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 and PCT / EP2017 / 056227 as originally filed. In yet another embodiment of the present invention, the antibody is an anti-TNFa antibody with a variable light chain domain and / or a variable domain. Petition 870250092396, dated 09 / 10 / 2025, p. 38 / 102 27 / 79 of the heavy chain comprising complementarity-determining regions (CDRs) with amino acid sequences, as disclosed in PCT applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 and PCT / EP2017 / 056227, as originally filed.

[00118] In a preferred embodiment of the present invention, the antibody is an anti-TNFa antibody with a variable light chain domain and / or a variable heavy chain domain comprising one or more CDRs with amino acid sequences, as disclosed in PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 or PCT / EP2017 / 056227. In another preferred embodiment of the present invention, the antibody is an anti-TNFa antibody with a variable light chain domain and a variable heavy chain domain comprising CDRs with amino acid sequences, as disclosed in claim 2 of PCT / EP2017 / 056218, claim 2 of PCT / EP2017 / 056246, claim 2 of PCT / EP2017 / 056237 or claim 2 of PCT / EP2017 / 056227, as originally filed.In yet another preferred embodiment of the present invention, the anti-TNFα antibody is selected from the group consisting of anti-TNFα antibodies comprising a variable domain heavy chain amino acid sequence and / or a variable domain light chain amino acid sequence according to claim 4 of PCT / EP2017 / 056218. Petition 870250092396, dated 09 / 10 / 2025, p. 39 / 102 28 / 79 claims 5 and 6 of PCT / EP2017 / 056246, claims 5 and 6 of PCT / EP2017 / 056237, claim 4 of PCT / EP2017 / 056227, and combinations thereof. The disclosure of each of the international patent applications PCT / EP2017 / 056218, PCT / EP2017 / 056246, PCT / EP2017 / 056237 and PCT / EP2017 / 056227 is incorporated herein in its entirety. They form part of the disclosure of this application.

[00119] In a specific embodiment, the antibody of the invention comprises (i) a VL domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 3, a CDR2 region with the amino acid sequence as shown in SEQ ID NO: 4, and a CDR3 region with the amino acid sequence as shown in SEQ ID NO: 5 and (ii) a VH domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 6, a CDR2 region with the amino acid sequence as shown in SEQ ID NO: 7, and a CDR3 region with the amino acid sequence as shown in SEQ ID NO: 8.

[00120] In a more preferred embodiment, the antibody of the invention comprises a VH domain with the amino acid sequence as shown in SEQ ID NO: 9. In another more preferred embodiment, the antibody comprises a VL domain with the amino acid sequence as shown in SEQ ID NO: 10. Most preferably, the antibody of the invention comprises (i) a VH domain with the sequence of Petition 870250092396, dated 09 / 10 / 2025, p. 40 / 102 29 / 79 amino acids as shown in SEQ ID NO: 9 and (ii) a Vl domain with the amino acid sequence as shown in SEQ ID NO: 10.

[00121] In another specific embodiment, the antibody of the invention comprises (i) a Vl domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 14, a CDR2 region with the amino acid sequence as shown in SEQ ID NO: 15, and a CDR3 region with the amino acid sequence as shown in SEQ ID NO: 16 and (ii) a Vh domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 17, a CDR2 region with the amino acid sequence as shown in SEQ ID NO: 18, and a CDR3 region with the amino acid sequence as shown in SEQ ID NO: 19.

[00122] In a more preferred embodiment, the antibody of the invention comprises a Vh domain with the amino acid sequence as shown in SEQ ID NO: 20. In another more preferred embodiment, the antibody comprises a Vl domain with the amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22. More preferably, the antibody of the invention comprises (i) a Vh domain with the amino acid sequence as shown in SEQ ID NO: 20 and (ii) a Vl domain with the amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22. Petition 870250092396, dated 09 / 10 / 2025, p. 41 / 102 30 / 79

[00123] The antibody of the invention has a high affinity for human TNFα. The term Kd refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibody of the invention binds to human TNFα with a dissociation equilibrium constant (KD) of less than approximately 2 x 10⁻¹⁰ M, preferably less than 1.5 x 10⁻¹⁰ M, preferably less than 1.25 x 10⁻¹⁰ M, more preferably less than 1 x 10⁻¹⁰ M, more preferably less than 7.5 x 10⁻¹¹ M or even less than 5 x 10⁻¹¹ M, as determined using surface plasmon resonance (SPR) technology on a BIACORE instrument. In particular, the determination of KD is performed as described in Example 1. Modifications that affect affinity with FcRn

[00124] The antibody of the invention comprises an amino acid sequence that differs from the native sequence of a wild-type antibody by virtue of at least one amino acid modification, as defined herein. The at least one amino acid modification affects the antibody's affinity for human FcRn. Typically, the at least one amino acid modification increases the antibody's affinity for human FcRn at pH 6. In one embodiment, the at least one amino acid modification increases the antibody's affinity for human FcRn at pH 6, wherein it does not substantially alter the affinity for human FcRn at pH 7.4. Preferably, Petition 870250092396, dated 09 / 10 / 2025, p. 42 / 102 31 / 79 The modified antibody has at least one amino acid substitution compared to the amino acid sequence of a wild-type antibody or a parental antibody, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions. Preferably, at least one amino acid modification is within the antibody's FcRn binding site. The antibody may have one or more amino acid modifications outside the antibody's FcRn binding site that affect FcRn binding, for example, by structural changes. The amino acid modification(s) may be generated by methods that are 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).

[00125] The antibody of the invention comprises the amino acid tryptophan at position 434 (EU numbering). This is referred to as 434W in this document. The native amino acid at position 434 of unmodified human IgG antibodies is asparagine (N). Thus, the antibody of the invention can be obtained by introducing the N434W mutation into an antibody. Preferably, the antibody of the invention is obtained or acquired by replacing asparagine with tryptophan at position 434. Petition 870250092396, dated 09 / 10 / 2025, page 43 / 102 32 / 79

[00126] The antibody of the invention comprises the amino acid glutamic acid at position 428 (EU numbering). This is referred to as 428E in this document. The native amino acid at position 428 of unmodified human IgG antibodies is methionine (M). Thus, the antibody of the invention can be obtained by introducing the M428E mutation into an antibody. Preferably, the antibody of the invention is obtainable or obtained by replacing methionine with glutamic acid at position 428.

[00127] Preferably, the antibody of the present invention further comprises the amino acid arginine at position 311 (EU numbering). This is referred to as 311R in this document. The native amino acid at position 311 of unmodified human IgG antibodies is glutamine (Q). Thus, the antibody of the invention can be obtained by introducing the Q311R mutation into an antibody. Preferably, the antibody of the invention is obtained or acquired by replacing glutamine with arginine at position 311.

[00128] In one embodiment, the antibody of the invention comprises amino acid 434W and amino acid 428E. In another embodiment, the antibody of the invention comprises amino acid 434W and amino acid 311R. In a preferred embodiment of the invention, the antibody comprises amino acids 434W, 428E, and 311R. This antibody is obtained by introducing Q311R, M428E, and N434W mutations into an antibody. Petition 870250092396, dated 09 / 10 / 2025, p. 44 / 102 33 / 79

[00129] The remaining amino acid sequence of the constant domain may be identical to the native amino acid sequence of a typical human IgG. It is possible, however, that the antibody amino acid sequence may comprise one or more additional mutations or substitutions from the native amino acid sequence of the Fc region of a native antibody, provided that the antibody still has TNFα binding activity and effector functions.

[00130] In a preferred embodiment, the Fc region of the antibody of the invention, including the hinge region, comprises or consists of the amino acid sequence as shown in SEQ ID NO: 13.

[00131] In one embodiment, the heavy chain of the antibody of the invention has the amino acid sequence as shown in SEQ ID NO: 11. Preferably, this antibody further comprises a light chain with the amino acid sequence as shown in SEQ ID NO: 1.

[00132] In another embodiment, the heavy chain of the antibody of the invention has the amino acid sequence as shown in SEQ ID NO: 12. Preferably, this antibody further comprises a light chain with the amino acid sequence as shown in SEQ ID NO: 23 or SEQ ID NO: 24.

[00133] In a preferred aspect of the invention, the antibody of the invention is a non-fucosylated antibody or an antibody with reduced fucosylation. Petition 870250092396, dated 09 / 10 / 2025, p. 45 / 102 34 / 79

[00134] The term reduced-fucosylation antibody, as used herein, refers to an antibody in which less than 90% of the antibody's N-glycans are fucosylated. Methods for determining the percentage of fucosylation are known in the art. Preferably, the percentage of fucosylation is determined as described in Example 11 of this application.

[00135] In one embodiment, less than 75%, or less than 50%, or less than 25% of the antibody N-glycans are fucosylated. More preferably, less than 15% of the antibody N-glycans are fucosylated. In a particular embodiment, the antibody N-glycans of the invention do not contain fucose.

[00136] Preferably, less than 90% of the N-glycans in 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 in N297 (EU numbering) of the antibody are fucosylated. More preferably, less than 15% of the N-glycans in N297 (EU numbering) of the antibody are fucosylated.

[00137] In another embodiment, the N-glycans in N297 of the antibody do not contain fucose.

[00138] Non-fucosylated antibodies, sometimes also called afucosylated antibodies, can be generated by various methods. For example, knockdown Petition 870250092396, dated 09 / 10 / 2025, page 46 / 102 Synergistic 35 / 79 of the α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) genes in CHO cells can be used to produce fully afucosylated and ADCC-enhanced monoclonal antibody variants (see, for example, Imai-Nishiya et al. (2007) BMC Biotechnol. 7, 84). A method using zinc finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of α1,6-fucosyltransferase and thus disrupt the corresponding enzymatic function in CHO cells can be used to produce completely fucose-free monoclonal antibodies (see, for example, Malphettes et al. (2010) Biotechnol. Bioeng. 106, 774-783).

[00139] Antibodies with reduced fucosylation can be prepared by adding a decoy substrate such as 2-deoxy-2-fluoro-2-fucose to the culture medium (see, for example, Dekker et al. (2016) Sci Rep 6: 36964), resulting in reduced incorporation of fucose into IgG-Fc glycans.

[00140] In another embodiment, the antibody of the invention has a high sialic acid content. It is possible to obtain an increase in sialylation, for example, by simultaneous transfection of cytidine monophosphate-sialic acid synthase (CMP-SAS), cytidine monophosphate-sialic acid (CMP-SAT) and α 2,3-sialyltransferases (see, for example, Son et al. (2011) Glycobiology 21, 1019-1028). Affinity with FcRn Petition 870250092396, dated 09 / 10 / 2025, page 47 / 102 36 / 79

[00141] The affinity at pH 6 for human FcRn of the antibody of the invention is high. The high-affinity binding of the antibody to human FcRn at pH 6 is characterized by a KD value of less than 100 nM. Preferably, the KD value of the high-affinity binding at pH 6 is less than 75 nM, or less than 50 nM, or less than 25 nM, or even less than 10 nM. For example, the KD value that characterizes the affinity for human FcRn at pH 6 may be in the range of 1 nM to 500 nM, or 2 nM to 100 nM, or 3 nM to 50 nM, or 4 nM to 25 nM, or 5 nM to 10 nM.

[00142] In a preferred embodiment, the affinity of the antibody of the invention to human FcRn at pH 6 is higher than the affinity of infliximab to human FcRn at pH 6.0.

[00143] The affinity of the antibody of the invention to human FcRn is preferably determined by surface plasma resonance (SPR), for example, as described in Example 4 of this application.

[00144] The antibody of the present invention typically has low affinity for human FcRn at pH 7.4. Low affinity is characterized by a Kd value greater than 1 pM. Preferably, low affinity for human FcRn at pH 7.4 is characterized by a Kd value greater than 2 pM, or greater than 5 pM, or greater than 10 pM. Petition 870250092396, dated 09 / 10 / 2025, p. 48 / 102 37 / 79

[00145] In a particular embodiment, the low affinity is so low that a Kd value cannot be determined by SPR.

[00146] In a special embodiment, the ratio of (i) a KD value for binding of the antibody of the invention to human FcRn at pH 7.4 to (ii) a KD value for binding to human FcRn at pH 6.0 is at least 100. Preferably, this ratio is at least 200 or at least 300 or at least 400 or at least 500 or at least 600 or at least 700 or at least 800 or at least 800 or at least 900 or at least 1000. Functional properties of the antibody

[00147] The antibody of the invention is efficiently transported through a monolayer of polarized cells from the apical to the basolateral side. Typically, transport through the monolayer of polarized cells is in a greater quantity than that of infliximab, wherein the amount of antibody in infliximab refers to the mass / cm² of the monolayer of polarized cells. The amount of antibody transported through the monolayer of polarized cells, relative to the amount of infliximab transported through the monolayer of polarized cells, is at least 110%, preferably at least 125%, more preferably at least 150% or at least 175% or Petition 870250092396, dated 09 / 10 / 2025, page 49 / 102 38 / 79 at least 200% (where the amount of infliximab transported is defined as 100%).

[00148] In addition, the antibody is specifically transported across the monolayer of polarized cells from the apical side to the basolateral side in the presence of an excess of competing immunoglobulins. This is referred to as specific transport in this document.

[00149] The percentage of the total mass of immunoglobulins transported across the polarized cell monolayer is greater than the percentage of infliximab transported across the polarized cell monolayer from the apical to the basolateral side in the presence of a 10-fold excess of competing immunoglobulins. The percentage of antibody of the invention transported across the polarized cell monolayer in the presence of a 10-fold excess of unrelated immunoglobulins, relative to the percentage of infliximab transported across the polarized cell monolayer in the presence of a 10-fold excess of unrelated antibodies, is at least 150% or at least 200% or at least 250% or at least 300% (infliximab is defined as 100%).

[00150] Preferably, the polarized cell monolayer is a polarized T84 cell monolayer. The transcytosis process mimicking the transport assay can be performed as described in Example 5 of this application. Petition 870250092396, dated 09 / 10 / 2025, p. 50 / 102 39 / 79

[00151] The antibody of the invention binds to CD64, CD32a (H), CD32a (R), CD32b, CD16a (V), CD16a (F) and CD16b (NA2).

[00152] The antibody of the invention typically binds to CD64 with a Kd less than 100 nM, preferably less than 10 nM.

[00153] The antibody of the invention typically binds to CD32a (H) with a Kd less than 10 μM.

[00154] The antibody of the invention typically binds to CD32a (R) with a Kd less than 10 μM.

[00155] The antibody of the invention typically binds to CD32b with a KD of less than 10 μM.

[00156] The antibody of the invention typically binds to CD16a (V), for example, with a Kd less than 500 nM, preferably less than 100 nM.

[00157] The antibody of the invention typically binds to CD16a (F), for example, with a Kd less than 10 μM, preferably less than 1 μM.

[00158] The antibody of the invention typically binds to CD16b (NA2), for example, with a Kd less than 10 μM, preferably less than 1 μM.

[00159] The antibody of the invention still binds to human C1q. Preferably, this binding is stronger than the binding of infliximab to human C1q.

[00160] The antibody of the invention also possesses complement-dependent cytotoxicity (CDC) of Petition 870250092396, dated 09 / 10 / 2025, p. 51 / 102 40 / 79 rabbit complement. This CDC of the antibody of the invention is preferably larger than that of infliximab, in terms of relative EC50 and / or relative maximum mortality.

[00161] The antibody of the invention is also capable of inducing CD14+CD206+ macrophages. The level of induction is preferably comparable to or greater than that of infliximab.

[00162] The antibody of the invention is also capable of suppressing T cell proliferation. The degree of suppression of T cell proliferation is preferably comparable to or greater than infliximab. Pharmaceutical Compositions and Treatment

[00163] The treatment of a disease encompasses the treatment of patients already diagnosed as having any form of the disease at any clinical stage or manifestation; delaying the onset or progression or worsening or deterioration of the symptoms or signs of the disease; and / or preventing and / or reducing the severity of the disease.

[00164] An individual or patient to whom an anti-TNFa antibody is administered may 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 respects, the human is a pediatric patient. In other respects, the human is an adult patient. Petition 870250092396, dated 09 / 10 / 2025, page 52 / 102 41 / 79

[00165] Compositions comprising an anti-TNFα antibody and, optionally, one or more additional therapeutic agents, such as the second therapeutic agents described below, are described herein. The compositions are typically supplied as part of a sterile pharmaceutical composition that includes a pharmaceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administration to a patient).

[00166] Anti-TNFα antibodies can be administered to a patient via a variety of routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intrathecally, topically, or locally, for example, via the mucosa. The most appropriate route of administration in any case will depend on the specific antibody, the individual, and the nature and severity of the disease and the individual's physical condition. Typically, an anti-TNFα antibody will be administered intravenously.

[00167] In a particularly preferred embodiment, the antibody of the invention is administered orally. If administered orally, the antibody is preferably an IgG, more preferably an IgG1.

[00168] In typical embodiments, an anti-TNFo antibody is present in a pharmaceutical composition at a concentration sufficient to permit administration. Petition 870250092396, dated 09 / 10 / 2025, p. 53 / 102 42 / 79 intravenously from 0.5 mg / kg of body weight to 20 mg / kg of body weight. In some embodiments, the antibody concentration suitable for use in the compositions and methods described herein includes, but is not limited to, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg or a concentration ranging between any of the preceding values, for example, from 1 mg / kg to 10 mg / kg, from 5 mg / kg to 15 mg / kg or from 10 mg / kg to 18 mg / kg.

[00169] The effective dose of an anti-TNFα antibody can range from about 0.001 to about 750 mg / kg per single administration (e.g., bolus), multiple administrations, or continuous administration, or to achieve a serum concentration of 0.01–5000 μg / ml per single dose (e.g., bolus), multiple administrations, or continuous administration, or any effective range or value, depending on the condition being treated, the route of administration, and the age, weight, and condition of the individual. In the case of oral administration, the serum concentration may be very low or even below the detection limit. In certain modalities, each dose may range from about 0.5 mg to about 50 mg per kilogram of body weight or from about 3 mg Petition 870250092396, dated 09 / 10 / 2025, p. 54 / 102 43 / 79 at approximately 30 mg per kilogram of body weight. The antibody can be formulated as an aqueous solution.

[00170] In a particularly preferred embodiment, the antibody of the invention is administered orally. If administered orally, the antibody is preferably an IgG, more preferably an IgG1. If the antibody is administered orally, the daily dose of antibody is typically in the range of about 0.01 mg / kg to about 100 mg / kg of body weight, or about 0.05 mg / kg to about 50 mg / kg of body weight, or about 0.1 mg / kg to about 25 mg / kg of body weight, or about 0.15 mg / kg to about 10 mg / kg of body weight, or about 0.16 mg / kg to about 5 mg / kg of body weight, or about 0.2 mg / kg to about 2 mg / kg of body weight, or about 0.2 mg / kg to about 1 mg / kg of body weight. Generally, beneficial doses are 1 to 200 mg per day, preferably 5 to 100 mg or 10 to 50 mg per day.

[00171] Pharmaceutical compositions may conveniently be presented in unit dose forms containing a predetermined amount of an anti-TNFα antibody per dose. This unit may contain from 0.5 mg to 5 g, for example, but without limitation, 1 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 200 mg, 200 mg, 300 mg, 400 mg, 500 mg, 750 mg, 1000 mg or any interval between two of the preceding values, for example from 10 mg to 1000 mg, from 20 mg to 50 mg Petition 870250092396, dated 09 / 10 / 2025, page 55 / 102 44 / 79 or 30 mg to 300 mg. Pharmaceutically acceptable carriers can take a wide variety of forms, depending, for example, on the condition being treated or the route of administration.

[00172] Determining the effective dosage, total number of doses, and duration of treatment with an anti-TNFα antibody is well within the capabilities of those skilled in the art and can be determined using a standard dose escalation study.

[00173] Therapeutic formulations of suitable anti-TNFA antibodies in the methods described in this document may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibody to the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers typically employed in the art (all of which are referred to herein as carriers), i.e., buffering agents, stabilizing agents, preservatives, isotonic agents, non-ionic detergents, antioxidants, and other miscellaneous additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). These additives must be non-toxic to the receptors at the dosages and concentrations employed.

[00174] Buffer agents help maintain pH in a range that approximates physiological conditions. They can have concentrations ranging from about 2 mM to about Petition 870250092396, dated 09 / 10 / 2025, page 56 / 102 45 / 79 of 50 mM. Suitable buffering agents include organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), citrate-phosphate buffers, succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-sodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, fumarate mixture monosodium fumarate, etc.), gluconate buffers (e.g., gluconic acid-sodium glycolate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glucosinolate mixture, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and buffers of. Petition 870250092396, dated 09 / 10 / 2025, page 57 / 102 46 / 79 acetate (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, may be used.

[00175] The pharmaceutical composition of the invention may further comprise at least one salt, for example, sodium chloride. The concentration of the salt preferably ranges from 100 mM to 200 mM, for example, about 150 mM.

[00176] Preservatives may be added to retard microbial growth and may be added in amounts ranging from 0.2% to 1% (w / v). Suitable preservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as stabilizers, may be added to ensure the isotonicity of the liquid compositions 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 range from bulking agents to solubilizing agents. Petition 870250092396, dated 09 / 10 / 2025, p. 58 / 102 47 / 79 therapeutic or helps prevent denaturation or adhesion to the container wall. Typical stabilizers may be polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; Disaccharides, such as lactose, maltose, sucrose, and trisaccharides, such as raffinose; and polysaccharides, such as dextran. Stabilizers may be present in the range of 0.1 to 10,000 wt. per pound of active protein.

[00177] Surfactants or non-ionic detergents (also known as wetting agents) can be Petition 870250092396, dated 09 / 10 / 2025, p. 59 / 102 48 / 79 added to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also allows the formulation to be exposed to surface shear under tension without causing protein denaturation. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxymers (184, 188, etc.), pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.). Non-ionic surfactants may be present in a range of about 0.05 mg / ml to about 1.0 mg / ml, or in a range of about 0.07 mg / ml to about 0.2 mg / ml.

[00178] Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), protease inhibitors, and co-solvents.

[00179] The formulation herein may also contain a second therapeutic agent in addition to an anti-TNFa antibody. Examples of suitable second therapeutic agents are provided below.

[00180] The dosing schedule may vary from once a month to daily, depending on several clinical factors, including the type of disease, the severity of the disease, and the patient's sensitivity to the anti-TNFa antibody. In specific modalities, an anti-TNFa antibody is Petition 870250092396, dated 09 / 10 / 2025, pages 60 / 102 49 / 79 administered daily, twice a week, three times a week, every two days, every 5 days, once a week, every 10 days, every two weeks, every three weeks, every four weeks, or once a month, or in any range between two of the previous values, for example, every four days every month, every 10 days every two weeks, or two to three times a week, etc.

[00181] The dosage of an anti-TNFa antibody to be administered will vary according to the specific antibody, the individual and the nature and severity of the disease, the individual's physical condition, the therapeutic regimen (e.g., if a second therapeutic agent is used) and the route of administration selected; the appropriate dosage can be readily determined by a person skilled in the art.

[00182] It will be recognized by a person skilled in the art that the ideal amount and spacing of individual doses of an anti-TNFa antibody will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular individual being treated, and that a physician will determine the appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop, the amount and / or frequency of the dosage may be altered or reduced, in accordance with normal clinical practice. Petition 870250092396, dated 09 / 10 / 2025, pp. 61 / 102 50 / 79 Disorders being treated

[00183] The invention relates to a method of treating or preventing a human TNFα-related disease in an individual, comprising administering to the individual the antibody as defined herein. The term TNFα-related disorder or TNFα-related disease refers to any disorder whose onset, progression or persistence of symptoms or disease states requires the participation of TNFα.Exemplary disorders related to TNFα include, but are not limited to, chronic and / or autoimmune inflammatory states in general, immune-mediated inflammatory disorders in general, inflammatory disease of the CNS, inflammatory diseases affecting the eyes, joints, skin, mucous membranes, central nerves, gastrointestinal system, urinary tract or lungs, uveitis states in general, retinitis, HLA-B27+ uveitis, Behçet's disease, dry eye syndrome, glaucoma, Sjögren's syndrome, diabetes mellitus (including diabetic neuropathy), insulin resistance, arthritis states in general, rheumatoid arthritis, osteoarthritis, reactive arthritis and Reiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, amyotrophic lateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidney disease, cystitis, psoriasis. arthritis), hidradenitis suppurativa, panniculitis, pyoderma gangrenosum, SAPHO syndrome (synovitis, acne, Petition 870250092396, dated 09 / 10 / 2025, pp. 62 / 102 51 / 79 pustule) ischemia, hyperostosis and osteitis), acne, Sweet's syndrome, pemphigus, Crohn's disease (including extra-intestinal manifestations), ulcerative colitis, bronchial asthma, hypersensitivity pneumonitis, general allergies, allergic rhinitis, allergic sinusitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, Wegener's granulomatosis, Kawasaki syndrome, giant cell arteritis, Churg-Strauss vasculitis, burns, graft-versus-host disease, host-versus-graft reactions, rejection episodes after organ or bone marrow transplantation, systemic and local vasculitis states in general, systemic and cutaneous lupus erythematosus, polymyositis and dermatomyositis, scleroderma, pre-eclampsia, acute and chronic pancreatitis, viral hepatitis, alcoholic hepatitis, post-surgical inflammation, such as after eye surgery (e.g., surgery of cataract (replacement of the eye lenses) or glaucoma), 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 intra-abdominal and gynecological procedures, endoscopic urological procedures (e.g., prostate surgery, ureteroscopy, cystoscopy, interstitial cystitis), or Petition 870250092396, dated 09 / 10 / 2025, pp. 63 / 102 52 / 79 Perioperative inflammation (prevention) in general, bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, pustular dermatitis, cerebral malaria, hemolytic-uremic syndrome, allograft rejection, otitis media, snake bite, erythema, myelodysplastic syndromes, primary sclerosing cholangitis, seronegative spondylarthopathy, autoimmune hematolytic anemia, orofacial granulomatosis, pyostomatitis vegetans, aphthous stomatitis, geographic tongue, migratory stomatitis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Bell's palsy, Creutzfeldt-Jakob disease, and neurodegenerative conditions in general.

[00184] Cancer-related osteolysis, cancer-related inflammation, cancer-related pain, cancer-related cachexia, bone metastases, acute and chronic forms of pain, regardless of whether they are caused by central or peripheral effects of TNFα and whether they are classified as inflammatory, nociceptive or neuropathic forms of pain, sciatica, low back pain, carpal tunnel syndrome, complex regional pain syndrome (CRPS), gout, postherpetic neuralgia, fibromyalgia, local pain states, chronic pain syndromes due to metastatic tumor, dysmenorrhea.

[00185] The specific disorders to be treated include arthritis in general, rheumatoid arthritis, Petition 870250092396, dated 09 / 10 / 2025, pp. 64 / 102 53 / 79 osteoarthritis, reactive arthritis, juvenile arthritis; psoriasis including psoriatic arthritis; inflammatory bowel disease, including Crohn's disease, ulcerative colitis including proctitis, sigmoiditis, proctosigmoiditis, left-sided colitis, extensive colitis and pancolitis, indeterminate colitis, microscopic colitis including collagen and lymphocytic colitis, colitis in connective tissue disease, divertic colitis, colitis in diverticular disease, eosinophilic colitis and pouchitis.

[00186] More preferably, the antibody of the invention is used to treat an inflammatory bowel disease, in particular Crohn's disease, ulcerative colitis or microscopic colitis. Crohn's disease may be upper ileal, colonic, ileocolonic or isolated (gastric, duodenal and / or jejunal) Crohn's disease, including non-stressful / non-penetrating, stressful, penetrating and perianal behavior, allowing any combination of location and behavior of the disease of any of the aforementioned. Ulcerative colitis may be ulcerative proctitis, proctosigmoiditis, left-sided colitis, panulcerative colitis and pouchitis. Combined therapy and other aspects

[00187] Preferably, the patient to be treated with an anti-TNFa antibody is also treated with another conventional medication. For example, a patient suffering from disease Petition 870250092396, dated 09 / 10 / 2025, pages 65 / 102 Inflammatory bowel disease (IBD), especially if moderate to severe, is typically treated with mesalazine or its derivatives or prodrugs, corticosteroids, for example, budesonide or prednisolone (oral or IV), immunosuppressants, for example, azathioprine / 6-mercaptopurine (6-MP) or methotrexate, cyclosporine, or tacrolimus. Other medications that may be co-administered to the patient include other anti-TNFα antibodies (e.g., infliximab, adalimumab, etanercept, certolizumab pegol, golimumab), integrin antagonists (e.g., natalizumab, vedolizumab), anti-IL-23 antibodies (e.g., MEDI2070), anti-p7 antibodies (e.g., etrolizumab), JAK inhibitors in the JAK / STAT pathway (e.g., tofacitinib), and others. Other medications that may be co-administered to the patient include immunosuppressants (e.g., azathioprine / 6-MP or methotrexate or oral cyclosporine) in order to maintain stable remission for a longer period.Yet another aspect of the invention is the use of an anti-TNF antibody as defined above to reduce inflammation.

[00188] Yet another aspect of the invention is an anti-TNF antibody as defined above for use in reducing inflammation in a patient suffering from an inflammatory condition. Petition 870250092396, dated 09 / 10 / 2025, pages 66 / 102 55 / 79

[00189] A further aspect of this invention is a method of treating an inflammatory condition, comprising administering to a patient in need an effective amount of an anti-TNFa antibody as defined above. The inflammatory condition is preferably one of the conditions described above.

[00190] A further aspect of this invention is a method of preventing an inflammatory condition, comprising administering to a patient in need an effective amount of an anti-TNFa antibody as defined above. The inflammatory condition is preferably one of the conditions described above.

[00191] Yet another aspect of the present invention is a method for improving the transcytosis of an antibody directed against TNFα, comprising introducing the Q311R, M428E and N434W substitutions into the amino acid sequence of the antibody, so as to obtain a modified antibody with enhanced transcytosis. The modified antibody is preferably an antibody as described above.

[00192] Yet another aspect of the present invention is a method for prolonging the plasma half-life of an antibody directed against TNFα, comprising introducing the Q311R, M428E and N434W substitutions into the amino acid sequence of the antibody, so as to obtain an antibody Petition 870250092396, dated 09 / 10 / 2025, p. 67 / 102 56 / 79 modified having a prolonged plasma half-life. The modified antibody is preferably an antibody as described above. The plasma half-life may be increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to the half-life of the unmodified antibody (i.e., the parent antibody without the Q311R, M428E, and N434W substitutions). Table 1. Overview of the sequences in the sequence listing. SEQ ID NO: Amino acid sequence description 1 Ab-wt light chain, the parent antibody of the modified antibodies used in the examples 2 Ab-wt heavy chain, the parent antibody of the modified antibodies used in the examples 3 CDR L1 of clone 16-22-H05 4 CDR L2 of clone 16-22-H05 5 CDR L3 of clone 16-22-H05 6 CDR H1 of clone 16-22-H05 7 CDR H2 of clone 16-22-H05 8 CDR H3 of clone 16-22-H05 9 Vh of humanized IgG of clone 16-22-H05 10 Vl of humanized IgG of clone 16-22-H05 11 Ab-REW heavy chain (based on clone 1622-H05) Petition 870250092396, dated 09 / 10 / 2025, pages 68 / 102 57 / 79 SEQ ID NO: Amino acid sequence description 12 Ab-REW heavy chain (based on clone 1722-B03) 13 Ab-REW Fc region (including hinge region) 14 CDR L1 of clone 17-22-B03 15 CDR L2 of clone 17-22-B03 16 CDR L3 of clone 17-22-B03 17 CDR H1 of clone 17-22-B03 18 CDR H2 of clone 17-22-B03 19 CDR H3 of clone 17-22-B03 20 Vh of humanized IgG of clone 17-22-B03 21 Vl of humanized IgG of clone 17-22-B03 (sc08) 22 Vl of humanized IgG of clone 17-22-B03 (sc02) 23 Humanized IgG light chain from clone 17-22-B03 (sc08) 24 Humanized IgG light chain from clone 17-22-B03 (sc02) Examples Antibody variants

[00193] Several variants of an anti-TNFa antibody (hereinafter referred to as the parent antibody or Ab-wt) have been generated by introducing substitutions in the Fc region of the antibody's amino acid sequence. The chain Petition 870250092396, dated 09 / 10 / 2025, pages 69 / 102 The 58 / 79 light chain of Ab-wt has the amino acid sequence as shown in SEQ ID NO: 1, and the heavy chain of Ab-wt has the amino acid sequence as shown in SEQ ID NO: 2. Mutations were introduced by site-directed mutagenesis using established methods. Briefly, mutations were introduced by PCR. The forward primer was designed to contain the intended mutation, while the reverse primer was designed so that the 5' ends of the two primers anneal side-by-side (but do not overlap) (Figure 9). PCR was performed for 25 cycles (98 °C for 10 s, 64 °C for 30 s, 72 °C for 3 min). Before running the PCR product on agarose gel, the unmutated PCR template was removed from the PCR product pool using the restriction enzyme DpnI. After gel purification of the PCR product, the blunt ends were ligated to obtain a circularized plasmid that was transformed into competent E. coli cells.After overnight incubation, several colonies were harvested, the plasmid DNA isolated and sequenced to confirm that the mutation had been incorporated.

[00194] Non-fucosylated variants were generated by adding 0.15 mM of the decoy substrate 2-deoxy-2-fluoro-2-fucose to the culture medium (Dekkers et al. (2016) Sci Rep 6: 36964). This resulted in a significantly reduced incorporation of fucose into IgG-Fc glano, as shown in Example 11 below. Petition 870250092396, dated 09 / 10 / 2025, pp. 70 / 102 59 / 79 Table 2: Antibody variants generated from an anti-TNFa antibody (EU numbering) Designation Mutations related to the parental antibody Ab-wt* None (= parental antibody) Ab-REW** Q311R / M428E / N434W Ab-REW-2FF** non-fucosylated variant of Ab-REW * Antibody not in accordance with the invention; ** Antibody in accordance with the invention

[00195] Ab-REW and Ab-REW-2FF antibodies are antibodies according to the present invention. Example 1. Affinity with TNFa Method:

[00196] Affinity with TNFa was measured by Biacore. A CM5 chip was prepared using standard Biacore amine immobilization procedures. After insertion of a 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 operating 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 bound response of ~1000 RU's in all 4 flow cells. To remove non-covalently bound material from all chip flow cells, Petition 870250092396, dated 09 / 10 / 2025, pp. 71 / 102Three 15-second washes of 50 mM NaOH were performed on 60 / 79. On the protein A chip, the antibody was captured in flow cells 2 and 4, with flow cells 1 and 3 used for reference subtraction. Study antibodies were diluted in PBS-T to 10 nM and injected at 2.5–7.5 pL to obtain 120 RU of captured antibody. The TNFα analyte was prepared at 500 μg / mL in water, as directed by the supplier, and subsequently diluted in PBS-T running buffer. Single-cycle kinetics were used to estimate steady-state affinity. For each single-cycle analysis, a titration of 5 analyte concentrations was injected onto the ligand, and then complex dissociation was measured. The surface was regenerated using glycine pH 1.7. A double-reference method was employed in which the data from the ligand-bound capture surface (fc 2 and 4) were subtracted from the reference surfaces where no ligand was captured (fc 1 and 3, respectively).Blank buffer injections were performed every 3 to 4 cycles and then subtracted from the analyte injection cycles to correct for minor changes in ligand capture surface area. Repeated analyte injections at the beginning and end of each analytical run were used to check for sample degradation or changes in instrument performance. All analysis was performed at 25 °C and the sample holder was incubated at 10 °C during runs. Petition 870250092396, dated 09 / 10 / 2025, pp. 72 / 102 61 / 79 experiments. Each experiment was performed at least three times. A 1-to-1 linkage model was used to fit the resulting kinetic data. Results:

[00197] All antibodies exhibited binding kinetics similar to TNFα, indicating that any introduced modification did not lead to significant changes in the antigen-binding region. For the Ab-REW-2FF antibody, the dissociation rate could not be measured, therefore the affinity (KD) could not be determined. However, the association rate was comparable to the other antibodies, indicating that the introduction of the mutation does not significantly affect binding. Table 3: Binding kinetics of human IgG1 variants to TNFα as determined by SPR. ka (106 / Ms) kd (10-5 / s) Kd (pM) Ab-wt 8.37± 0.11 3.45± 0.20 4, 13± 0, 19 Ab-REW 6.22± 0.91 2.04± 0.52 3.30± 0.74 Ab-REW-2FF 5.80± 0.58 nd* nd* * It was not possible to determine the dissociation rate (kd). Example 2. Power Method:

[00198] L929 cells were incubated with 0.25 ng / mL of TNFα and 1 μg / well of actinomycin D in the presence of serial dilutions of anti-TNFα antibody variants. Petition 870250092396, dated 09 / 10 / 2025, pp. 73 / 102 62 / 79 After incubation for 20 h at 37 °C / 5% CO2, proliferative responses were 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 formazan product by dehydrogenase enzymes present in metabolically active cells. The amount of formazan product measured by absorbance at 492 nm was directly proportional to the number of live cells in culture. Results:

[00199] The results are shown in Figure 1. The introduction of mutations in the Fc region of the anti-TNFa antibody did not affect potency. Example 3. Affinity for Fcy receptors (CD64, CD32a, CD32b, CD16a, CD16b) Method:

[00200] Affinity for FcyRs was measured by Biacore. A CM5 chip was prepared using standard Biacore amine immobilization procedures. After insertion of a 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 (PBS-T) buffer; prior to immobilization, the chip surface was primed with three injections of 50 mM NaOH. FcyRs were immobilized on Petition 870250092396, dated 09 / 10 / 2025, pp. 74 / 102 63 / 79 chip surface using a His-tag capture system. The anti-His tag chip was prepared according to the Biacore kit instructions, with ~12000 RU of antibody deposited on all 4 flow cells. To remove non-covalently bound material from all chip flow cells, three 30-second washes of 10 mM glycine, pH 1.5, were performed. Fcy receptors were diluted in PBS-T to a range of 0.5–2 μg / mL, with 2.5–5.0 μL injected into the chip, generating capture levels between 60 and 200 RUs. Antibodies were diluted in PBS-T prior to analysis. Single-cycle kinetics were used to estimate steady-state affinity. For each single-cycle analysis, a titration of 5 antibody concentrations was injected onto the FcyR ligand, and then the dissociation of the complex was measured. The surface was regenerated using the recommended solution, 10 mM glycine, pH 1.5, for anti-His capture surfaces.A double-reference method was employed in which ligand-bound capture surface data (fc 2 and 4) were subtracted from reference surfaces where no ligand was captured (fc 1 and 3, respectively). Blank buffer injections were performed for each antibody titration cycle and then subtracted from analyte injection cycles to correct for minor changes in ligand capture surface. All analysis was performed at 25 °C and the support was... Petition 870250092396, dated 09 / 10 / 2025, pp. 75 / 102 64 / 79 samples were incubated at 10 °C during the experimental runs. Each experiment was performed at least three times. Results:

[00201] Binding to CD64 was not affected by the manipulated anti-TNFa antibodies. The introduction of mutations did not affect the affinity for CD32a (H), CD32a (R), and CD32b. However, Ab-REW-2FF showed a 5.5-fold increase in affinity for CD16a (V). The non-fucosylated antibody Ab-REW2FF also had improved binding to the low-affinity CD16a and CD16b receptors. Table 4: Affinity for Fcy receptors CD64, CD32a (H), CD32a (R), and CD32b as determined by SPR. The mean affinity and standard deviation calculated from two or more independent experiments are shown. Affinity (Kd) CD64 CD32a(H) (pM) CD32a(R) (pM) CD32b (pM) (nM) Ab-wt 2.92 ± 0.07 0.67 ± 0.04 nd 3.14 ± 0.80 Ab-REW 2.80 ± 0.24 0.78 ± 0.05 1.60 ± 0.01 1.21 ± 0.32 Ab-REW-2FF 2.93 ± 0.16 1.87 ± 0.37 1.45 ± 0.01 1.13 ± 0.25 Table 5: Affinity for Fcy receptors CD16a (V), CD16a (F), and CD16b as determined by SPR. The mean affinity and standard deviation calculated from two or more independent experiments are shown. Petition 870250092396, dated 09 / 10 / 2025, pp. 76 / 102 65 / 79 Affinity (Kd) CD16a(V) (nM) CD16a(F) (μΜ) CD16b(NA2) (μΜ) Ab-wt 184 ± 31.9 nd > 3.00 Ab-REW 280 ± 36.1 2.41 ± 0.68 1.65 ± 0.36 Ab-REW-2FF 33.5 ± 0.99 0.15 ± 0.01 0.40 ± 0.07 Example 4. Affinity with FcRn Method:

[00202] SPR was performed using a Biacore 3000 instrument with CM5 sensor chips coupled to anti-TNFa IgG1 antibodies (~500 resonance units (RU)) using amine coupling chemistry as described by the manufacturer. Coupling was performed by injecting 2.0 pg / mL of each protein into 10 mM sodium acetate, pH 4.5, using the amine coupling kit (GE Healthcare). HBS-P buffer pH 7.4 (10 mM HEPES, 150 mM NaCl, 0.005% P20 surfactant) or phosphate buffer pH 6.0 (67 nM phosphate buffer, 150 mM NaCl, 0.005% Tween 20) were used as running and dilution buffers. Binding kinetics were determined by injecting titrated amounts (1000 - 31.2 nM) of Human FcRn labeled with monomeric His (hFcRn) on antibodies immobilized at pH 7.4 or pH 6.0. All SPR experiments were conducted at 25 °C with a flow rate of 40 µL / min. Binding data were set to zero and the reference cell value subtracted. The 1:1 Langmuir ligand binding model provided by the software was used. Petition 870250092396, dated 09 / 10 / 2025, pp. 77 / 102 66 / 79 BIAevaluation (version 4.1) was used to determine the binding kinetics. Results:

[00203] The results showed that the wild-type antibody Ab-wt bound strictly to hFcRn in a pH-dependent manner. All engineered antibody variants had a higher affinity for FcRn at pH 6.0, but maintained their pH dependence and did not bind to the receptor at pH 7.4. Surprisingly, the REW-containing variants showed >160 times stronger binding at acidic pH and yet no detectable binding under tested conditions at neutral pH. The antibody variants showed improved binding to FcRn compared to infliximab containing a wild-type IgG1 Fc region. Table 6: Affinity of antiTNFa antibody variants for FcRn at pH 6.0 and pH 7.4, as determined by SPR. pH 6.0 pH 7.4 Kd (nM) Change in weight times Change in IFX times Kd (nM) Ab-wt 1000 NA Ab-REW 5.61 178 75.8 NA Ab-REW-2FF 6.24 160 68.1 NA IFX 425 NA NA: Not acquired due to weak connection. Petition 870250092396, dated 09 / 10 / 2025, pp. 78 / 102 67 / 79 Example 5. Transcytosis Method:

[00204] Transwell filters (1.12 cm2) with collagen-coated polytetrafluoroethylene (PTFE) membranes with a pore size of 0.4 μm were incubated O / N in whole growth medium followed by seeding of 1.0 x 106 T84 cells per well. Transepithelial electrical resistance (TEER) was monitored daily using a MILLICELL-ERS-2 volt-ohm meter. Cultures were grown for 4-5 days before reaching confluence with a TEER value of ~1000-1300 Ω x cm2. Before the experiments, the monolayers were nutrient-free for 1 h in Hank's Balanced Salt Solution (HBSS). Then, 400 nM of the antibody variants or IFX alone or together with 4000 nM of human myeloma IgG with irrelevant specificity were added to the apical chamber of Transwell. Samples were collected from the basolateral reservoir at 0 and 4 h after addition. Antibody concentrations in the basolateral reservoir were determined by ELISA.In summary, 96-well Maxisorp plates were coated with O / N containing recombinant TNFα or a goat anti-human Fc-specific antibody, both diluted to 1 μg / ml in PBS. Subsequently, the plates were blocked with PBS containing 4% skim milk for 2 h at room temperature, followed by washing 4 times with PBS. Petition 870250092396, dated 09 / 10 / 2025, pp. 79 / 102 68 / 79 containing 0.05% Tween 20. Samples collected during transcytosis experiments were added to wells and incubated for 2 h at room temperature before washing as described above. Captured antibody variants, IFX or total IgG, were detected using a goat alkaline phosphatase (ALP)-conjugated anti-human Fc-specific antibody. Binding was visualized by adding 100 μL of ALP substrate, and the 405 nm absorption spectrum was recorded. The amount of transported antibody variants, IFX and total IgG, was calculated from standard curves of each of the individual antibody variants. Transcytosis of antibody variants through polarized human epithelial cells Results:

[00205] The engineered anti-TNFα antibody variants were tested for transcytosis through a cell monolayer and compared to the antibody by weight or IFX with another human anti-TNFα IgG1 antibody. The results are shown in Figure 2. The anti-TNFα antibody by weight was transported from the apical to the basolateral reservoir. Compared to IFX, another IgG1 antibody with an Ft region by weight, 2.8 times more Ab-REW was transported, which was also the case for Ab-REW-2FF. Petition 870250092396, dated 09 / 10 / 2025, pages 80 / 102 69 / 79 Transcytosis of antibody variants through polarized human epithelial cells in the presence of competitive IgG. Results:

[00206] The total amount of immunoglobulin transported across a polarized T84 cell monolayer from the apical to the basolateral reservoir when anti-TNFα antibody variants were incubated with a 10-fold excess of human myeloma IgG 4 hours after addition was comparable for all antibodies. However, an 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 a competing excess of human IgG with irrelevant specificity. The results are shown in Figure 3. Example 6. ADCC Method:

[00207] A Promega ADCC reporter bioassay master kit was used. Briefly, mTNFa CHO-K1 target cells at 1 x 10⁵ / mL were seeded in white (light background) tissue culture plates, 100 μL per well. Plates were incubated O / N at 37 °C / 5% CO₂. On day 2, 95 μL of assay medium were removed and replaced with 25 μL of Jurkat effector cells manipulated at 3 x 10⁶ / mL. The plates were then incubated for 6 hours. Petition 870250092396, dated 09 / 10 / 2025, pages 81 / 102 70 / 79 °C / 5% CO2. The BioGlo™ reagent was prepared at the end of the incubation. Plates were equilibrated at RT for 10–20 minutes before adding 75 μL of BioGlo™ reagent per well. After 5–10 minutes of incubation in the dark, luminescence was measured. A 4 PL model was used to fit the data. Results:

[00208] The results (see Figure 4) showed that all anti-TNFa antibodies induced ADCC, but with distinct strengths. Compared with the wild-type antibody Abwt, the antibody variants showed an increase in ADCC. Specifically, the non-fucosylated antibody variant AbREW-2FF significantly improved ADCC. Example 7. C1q bond Method:

[00209] ELISA was performed using 96-well MaxiSorp plates, where the wells were coated with human TNFα diluted to 1 μg / mL in PBS. After O / N incubation at 4 °C, the plates were blocked with PBS containing 4% skim milk for 1 h and washed four times with PBS containing 0.05% Tween20 (PBS-T). Then, titrated amounts of anti-TNFα IgG antibodies were diluted in PBS-T, added, and incubated for 1 h at room temperature. After washing with PBS-T, human C1q (0.5 pg / mL) was diluted in 0.1 M Veronal buffer (0.25 mM CaCl2 and 0.8 mM MgCl2, pH Petition 870250092396, dated 09 / 10 / 2025, pages 82 / 102 71 / 79 7.2), added to the wells and incubated for 1 h. Subsequently, the wells were washed as above before rabbit anti-human C1q diluted 1:5000 in PBS-T was added to the wells and incubated for 1 h. After washing, donkey HRP-conjugated rabbit anti-IgG diluted 1:5000 in PBS-T was added. Subsequently, the wells were washed and 100 μL of 3,3',5,5'-Tetramethylbenzidine was added to each well. Absorbance was measured at 620 nm using a Sunrise spectrophotometer. Results:

[00210] Variants of the anti-TNFa antibody were captured in human TNFa prior to the addition of human C1q. The results (see Figure 5) showed that the antibodies bound to C1q, but with distinct binding strengths. Specifically, Ab-REW and Ab-REW-2FF bound slightly stronger than IFX. The presence of fucose in the biantenna N-glucan bound to N2 97 had no or only a small influence on binding. The binding hierarchy from strongest to weakest was as follows: Ab-REW > Ab-REW-2FF > IFX. Example 8. CDC Method:

[00211] The anti-TNFα CDC assay measured antibody-dependent complement cytotoxicity. Target cells expressing mTNFα were seeded in microplates in the presence of anti-TNFα antibodies that promoted the potential Petition 870250092396, dated 09 / 10 / 2025, pp. 83 / 102 72 / 79 cytotoxic complement. Six independent sample replicates (anti-TNFa antibody variants) and 4 replicates of the reference standard (IFX) were prepared at 60 μg / mL, serially diluted (1.3-fold dilution steps), and the dilution plates sealed until use. Target cells containing a LUC viability reporter were prepared to 1.5 x 10⁵ / mL and stored in a water bath at 37 °C. Rabbit complement was diluted to a final assay concentration of 3-fold in DMEM High Glucose. Immediately after preparation, complement was combined with target cells in a 1:1 (v / v) ratio. Forty mL of the complement / target cell preparation were transferred to each well of the assay plate. Antibodies prepared on the dilution plate were transferred to cells (20 μL / well) and the plate was then incubated at 36 °C / 1% CO2 for 3.5 h. The assay plates were equilibrated at room temperature in the dark for 35 min.The constant Glo, which was equilibrated at room temperature for 120 minutes before use, was added to the assay plate (20 μL / well) and stored at room temperature in the dark for 35 minutes before luminescence measurement. The percentage of cell death was then calculated for each concentration of each sample, and a 4 PL model was used to fit the data. Results: Petition 870250092396, dated 09 / 10 / 2025, pages 84 / 102 73 / 79

[00212] The results are shown in Figure 6 and Table 7. The relative EC50 and maximum relative mortality % results for the test sample performance in the CDC assay provided a clear classification of activity that was consistent across both activity measures. Ab-REW showed higher CDC activity than IFX, regardless of fucose content. In a direct comparison between fucose variant samples, to better understand the impact of fucose content on CDC activity, Ab-REW-2FF compared to Ab-REW provided similar responses to CDC activity. The comparison showed a relative EC50 response of 97.1% and a maximum relative mortality response of 100.4%. Table 7: CDC activity of anti-TNFa antibody variants in terms of relative EC50 and % relative death performance compared to IFX. Sample ID EC50 relative (%) Maximum relative death (%) IFX 100 100 Ab-REW 123.7 113.8 Ab-REW-2FF 120.1 114.2 Example 9. Induction of regulatory macrophages Method:

[00213] Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coatings Petition 870250092396, dated 09 / 10 / 2025, pages 85 / 102 4 / 79 healthy cells were isolated by Ficoll gradient centrifugation. Cells from two individual donors were mixed in equal numbers, and 2 x 10⁵ cells from the mixture were plated in 96-well plates in a total volume of 100 pL / well. The cells were incubated for 48 hours at 37 °C / 5% CO₂. After 48 h, anti-TNFa or IFX antibody variants were added to achieve a final concentration of 10 pg / mL. Each compound was added in replicates of five or six. The final volume was 150 pL / well. Human IgG1 serum (Sigma # I5154) was used as a control. After the addition of the compounds, mixed lymphocyte reactions (MLRs) were cultured for a further 4 days at 37 °C / 5% CO₂. Subsequently, the plates were washed with PBS / 5 mM EDTA (PBS / EDTA) and incubated with 50 μL / well of PBS / EDTA for 20 min at room temperature. The plates were centrifuged and the liquid was removed.The antibody was diluted in PBS / EDTA (anti-CD14-PE, anti-CD206-APC, both diluted 1:10). Cells were resuspended in 50 μL of the antibody solution and incubated for 20 min at room temperature. Subsequently, the cells were washed with PBS / EDTA and resuspended in 50 mL of PBS / EDTA. The stained samples were analyzed on a Fortessa FACS using FACSDiva software. The analysis was performed using the software. FlowJo. Results: Petition 870250092396, dated 09 / 10 / 2025, pages 86 / 102 75 / 79

[00214] The induction of regulatory macrophages was analyzed in four independent MLRs and was successful in all experiments (comparing the IFX control to IgG). The results are shown in Figure 7. The levels of induction by IFX may differ between experiments due to the fact that each experiment was performed using different donors with interindividual variation. All anti-TNFα antibody variants tested induced CD14+CD206+ regulatory macrophages with slight variation between compounds. Ab-REW and Ab-REW-2FF induced slightly more regulatory macrophages than IFX; however, only in the case of Ab-REW-2FF was the increase significant. Example 10. Inhibition of T cell proliferation Method:

[00215] PBMCs were isolated from healthy buffy coats. Cells were isolated by Ficoll gradient centrifugation. Cells from two individual donors were mixed in equal numbers, and 2 x 10⁵ cells from the mixture were plated in 96-well plates at a total volume of 100 pL / well. Cells were incubated for 48 hours at 37 °C / 5% CO₂. After 48 h, anti-TNFa or IFX antibody variants were added to achieve a final concentration of 10 pg / mL. Each compound was added in replicates of five or six. The final volume was 150 pL / well. Human IgG1 serum (Sigma # I5154) was used. Petition 870250092396, dated 09 / 10 / 2025, pages 87 / 102 76 / 79 as a control. After the addition of the compounds, mixed lymphocyte reactions (MLRs) were cultured for a further 2 days at 37 °C / 5% CO2. Subsequently, tritiated thymidine (3H thymidine, 0.5 microCurie / well) was added to the cultures. The cultures were then incubated for 18 ha at 37 °C / 5% CO2. Samples were collected using a Microbeta Filtermat 96 cell collector 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). Results:

[00216] Inhibition of T cell proliferation was measured in three independent MLRs and was defined as successful if IFX as a positive control induced suppression. The levels of suppression by IFX in individual experiments may differ presumably due to variation in macrophage regulatory induction. In each experiment, the potential of the anti-TNFα antibody variants to suppress T cell proliferation was calculated relative to the positive control IFX. The Ab-REW-2FF antibody showed significantly enhanced suppression compared to IFX, while suppression by Ab-REW was comparable to IFX (see Figure 8). Example 11. Analysis of N-glycans Method: Petition 870250092396, dated 09 / 10 / 2025, pages 88 / 102 77 / 79

[00217] 50 µl of each IgG variant (1 mg / ml) were centrifuged for 10 min at 13,000 χ g before 1 pg of trypsin dissolved in 100 µl of 50 mM ammonium bicarbonate (pH 7.8) was added and incubated overnight at 37 °C. The centrifuges were centrifuged at 13,000 χ²g for 10 min, and the flow was transferred to an Eppendorf tube and dried in a SpeedVac (Heto Maxi dry). The dried samples were dissolved in 20 µl of 1% formic acid, sonicated for 30 min, and centrifuged for 10 min at 16,100 χ²g. Subsequently, each sample was transferred to new vials, and the analysis by reversed-phase inline liquid chromatography (C18) tandem liquid chromatography (LCMS / MS) of proteolytic peptides was performed using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, USA). 5 µl of peptide solution were injected into the extraction column, and the peptides were eluted in backwash mode from the extraction column to the analytical column. The mobile phase consists of acetonitrile and water for mass spectrometry, both containing 0.1% formic acid.Chromatographic separation was achieved using a binary gradient of 3 to 50% acetonitrile in water for 60 minutes at a flow rate of 0.3 µl / min. The LC system was coupled via a sprayed nano-ion source to an exact Q hybrid quadrupole orbiting mass spectrometer (Thermo Fisher Scientific, USA). The peptide samples... Petition 870250092396, dated 09 / 10 / 2025, pages 89 / 102 78 / 79 were analyzed using a high-energy collisional dissociation (HCD) fragmentation method with collision energy normalized to 20, acquiring an Orbitrap scan in the m / z 300-2000 mass range followed by MS / MS of the ten most intense ions in the Orbitrap.

[00218] Data analysis was performed on Xcalibur v2.0. MS / MS spectra for all N-glycopeptides were extracted by searching for oxonium ions; 204.086 (N-acetylhexosamine) and 366.1388 (N-acetylhexosamine-hexose). Using HCD fragmentation with collision energy normalized to 20, the glycan structure and peptide mass for IgG were detected. Extracted ion chromatograms for the target glycol peptides (EEQYNSTYR for IgG1) were extracted with an accuracy of 10 ppm and the corresponding MS / MS spectra were manually verified. HCD fragmentation with collision energy normalized to 35 was used to detect the peptide sequence and verify that the peptide mass corresponded to the correct peptide sequence. The area under the curve for all extracted glycol peptides was calculated, and the percentage ratio for each glycoform was determined. Results:

[00219] For Ab-REW, two forms of N-glycan dominated and accounted for > 90% of the total N-glycan pool, namely 4GlcNac-1Fuc-3Man and 4GlcNac-1Fuc-3Man Petition 870250092396, dated 09 / 10 / 2025, pages 90 / 102 79 / 79 1Gal. Both dominant N-glycan forms contained a fucose core. To produce the non-fucosylated version of Ab-REW (Ab-REW-2FF), the decoy substrate 2-deoxy-2-fluoro-1-fucose (2FF) was used. MS mapping of this antibody revealed that this strategy successfully resulted in a significantly reduced incorporation of fucose, with a drop from >90% to 13% detected. The dominant N-glycan forms after treatment were the same as the variants produced in the absence of 2FF, except that these structures lacked fucose (see also Figure 10). Table 8: Percentage of N-glycan forms bound to N297 of anti-TNFa IgG antibodies Structure of N- glycan Ab-REW (%) Ab-REW2FF (%) 4GlcNac-1Fuc-3Man 69.1 8.5 4GlcNac-1Fuc-3Man- 1Gal 21.7 4.5 4GlcNac-3Man 0.7 64.3 Nac-3G 17.3 Petition 870250092396, of 09 / 10 / 2025, p. 91 / 102

Claims

1 / 3 CLAIMS 1. An IgGi antibody characterized in that it comprises a TNFα-binding domain and an FcRn-binding site, having a high affinity for human FcRn at pH 6, said high affinity being defined by a dissociation equilibrium constant (KD), determined by surface plasmon resonance, of less than 100 nM, and yet having no or low affinity for human FcRn at pH 7.4, said low affinity being defined by a KD, determined by surface plasmon resonance, greater than 10 μM, wherein the amino acid sequence of the antibody comprises amino acids 311R, 428E and 434W, as defined by EU numbering, and wherein the antibody comprises (i) a Vl domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 3, a CDR2 region with the amino acid sequence as shown in SEQ ID NO: 4, and a region CDR3 with the amino acid sequence as shown in SEQ ID NO: 5,and (ii) a Vh domain comprising a CDR1 region with the amino acid sequence as shown in SEQ ID NO: 6, a CDR2 region with the amino acid sequence as shown in SEQ ID NO: 7, and a CDR3 region with the amino acid sequence as shown in SEQ ID NO:

8. Petition 870250092396, dated 09 / 10 / 2025, pp. 92 / 102 2 / 3, 2. IgG1 antibody, according to claim 1, characterized in that it is a non-fucosylated antibody or an antibody with reduced fucosylation.

3. IgG1 antibody, according to claim 1 or 2, characterized in that it comprises a heavy chain comprising the amino acid sequence as shown in SEQ ID NO:

13.

4. IgG1 antibody, according to any one of claims 1 to 3, characterized in that it comprises a VH domain with the amino acid sequence as shown in SEQ ID NO: 9 and a Vl domain having an amino acid sequence as shown in SEQ ID NO:

10.

5. IgG1 antibody, according to any one of claims 1 to 4, characterized in that it comprises a light chain with the amino acid sequence as shown in SEQ ID NO: 1 and a heavy chain with an amino acid sequence as shown in SEQ ID NO:

11.

6. Pharmaceutical composition characterized in that it comprises the antibody, as defined in either claim 1 or 5.

7. Use of an antibody, as defined in any one of claims 1 to 5, characterized by being for the preparation of a medicament for the treatment of a disease. Petition 870250092396, dated 09 / 10 / 2025, pp. 93 / 102 3 / 3 8. Use of an antibody, as defined in any one of claims 1 to 5, characterized in that it is for the preparation of a medicament for the treatment of an inflammatory condition.

9. Use, according to claim 8, characterized in that the inflammatory condition is an inflammatory disorder of the gastrointestinal tract.

10. Use, according to claim 8 or 9, characterized in that the said antibody of the medicament is prepared to be applied orally or topically. Petition 870250092396, dated 09 / 10 / 2025, pp. 94 / 102

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