Antibodies targeting TNF alpha and IL-23 and uses thereof

Abstract

Disclosed herein are dual specific antibodies that target both human TNF [alpha] and the P19 subunit of human IL-23. Also provided herein are polynucleotides encoding the dual specific antibodies, pharmaceutical compositions comprising the dual specific antibodies, and methods of producing the dual specific antibodies. Medical uses of the dual specific antibodies described herein are also disclosed.

Description

Technical Field

[0001] The present disclosure relates to molecular biology and immunology. Provided herein are bispecific antibodies that target both human TNFα (TNFα) and the P19 subunit of human IL-23 (IL23p19), and their use in treating autoimmune and inflammatory diseases. Background Art

[0002] Autoimmune and inflammatory diseases can be caused by the generation of an immune response by the body against its own tissues, and they are generally chronic and can be debilitating and even life-threatening. TNFα is a potent inducer of the inflammatory response, and interleukin 23 (IL-23) is an upstream regulator in tissue inflammation. Both TNFα and IL-23 are clinically validated targets. However, for patients who show an inadequate response to available therapeutic agents targeting TNFα or IL-23, the need for alternative treatment options remains largely unmet. The bispecific antibodies targeting both human TNFα and human IL23p19 and related methods provided herein meet these needs and provide relative benefits. Summary of the Invention

[0003] Provided herein are bispecific antibodies that specifically bind to human TNFα (TNFα) and bind to the P19 subunit of human IL-23 (IL23p19), and related pharmaceutical compositions, polynucleotides, vectors, and cells. Also provided herein are methods for generating these bispecific antibodies and using these bispecific antibodies. Some exemplary embodiments are provided below.

[0004] Embodiment 1: A bispecific antibody that specifically binds to human TNFα and binds to human IL23p19, comprising (1) a first light chain (LC1) comprising a first light chain variable domain (VL1) and a first heavy chain constant domain 1 (CH1); (2) a first heavy chain (HC1) comprising a first heavy chain variable domain (VH1), a first light chain constant region (CL), and a Knob-Fc region; (3) a second light chain (LC2) comprising a second light chain variable domain (VL2) and a second CL region; and (4) a second heavy chain (HC2) comprising a second heavy chain variable domain (VH2), a second CH1 domain, and a Hole-Fc region; wherein (i) the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively; and (ii) the Knob-Fc region is a variant of the human IgG Fc region having a T366W substitution (according to EU index numbering); and the Hole-Fc region is a variant of the human IgG Fc region having a Y407V substitution (according to EU index numbering).

[0005] Embodiment 2: The bispecific antibody according to Embodiment 1, wherein (1) the first CL region is kappa CL (Cκ; SEQ ID NO: 68) or lambda CL (Cλ, SEQ ID NO: 69); and the second CL region is Cκ (SEQ ID NO: 68) or Cλ (SEQ ID NO: 69); (2) both the first CH1 domain and the second CH1 domain are human IgG1 CH1 domains (SEQ ID NO: 61); or (3) the pestle-Fc region has the amino acid sequence shown in SEQ ID NO: 66; and the mortar-Fc region has the amino acid sequence shown in SEQ ID NO: 67; or any combination of (1)-(3).

[0006] Embodiment 3: The bispecific antibody according to Embodiment 1 or 2, wherein the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19.

[0007] Embodiment 4: The bispecific antibody according to Embodiment 3, wherein VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 5 and 6 respectively; or wherein VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 14 and 15 respectively.

[0008] Embodiment 5: The bispecific antibody according to Embodiment 3, wherein VL1, VH1, VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 1, 2, 10 and 11 respectively; (2) the amino acid sequences shown by SEQ ID NO: 1, 2, 14 and 15 respectively; (3) the amino acid sequences shown by SEQ ID NO: 5, 6, 10 and 11 respectively; or (4) the amino acid sequences shown by SEQ ID NO: 5, 6, 14 and 15 respectively.

[0009] Embodiment 6: The bispecific antibody according to Embodiment 3, wherein LC1, HC1, LC2 and HC2 have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 24, 25, 12 and 26 respectively; (2) SEQ ID NO: 27, 28, 12 and 26 respectively; (3) SEQ ID NO: 24, 25, 16 and 29 respectively; or (4) SEQ ID NO: 27, 28, 16 and 29 respectively.

[0010] Embodiment 7: The bispecific antibody according to Embodiment 1 or 2, wherein the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα.

[0011] Embodiment 8: The bispecific antibody according to Embodiment 7, wherein VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO:10 and 11 respectively; or (2) the amino acid sequences shown by SEQ ID NO:14 and 15 respectively; or wherein VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO:1 and 2 respectively; or (2) the amino acid sequences shown by SEQ ID NO:5 and 6 respectively.

[0012] Embodiment 9: The bispecific antibody according to Embodiment 7, wherein VL1, VH1, VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO:10, 11, 1 and 2 respectively; (2) the amino acid sequences shown by SEQ ID NO:10, 11, 5 and 6 respectively; (3) the amino acid sequences shown by SEQ ID NO:14, 15, 1 and 2 respectively; or (4) the amino acid sequences shown by SEQ ID NO:14, 15, 5 and 6 respectively.

[0013] Embodiment 10: The bispecific antibody according to Embodiment 7, wherein LC1, HC1, LC2 and HC2 have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO:18, 19, 3 and 20 respectively; (2) SEQ ID NO:21, 22, 3 and 20 respectively; (3) SEQ ID NO:18, 19, 7 and 23 respectively; or (4) SEQ ID NO:21, 22, 7 and 23 respectively.

[0014] Embodiment 11: A bispecific antibody that specifically binds to human TNFα and binds to human IL23p19, comprising (1) a light chain (LC) that comprises a first light chain variable domain (VL1) and a CL region; and (2) a heavy chain (HC) that comprises a first heavy chain variable domain (VH1), a heavy chain constant region (CH), a second light chain variable domain (VL2); and a second heavy chain variable domain (VH2); wherein the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19 respectively, or specifically bind to human IL23p19 and human TNFα respectively.

[0015] Embodiment 12: The bispecific antibody according to Embodiment 11, wherein (1) the CL region is Cκ (SEQ ID NO: 68) or Cλ (SEQ ID NO: 69); or (2) the CH region is a human IgG1 CH region (SEQ ID NO: 58); or both (1) and (2).

[0016] Embodiment 13: The bispecific antibody according to Embodiment 11 or 12, wherein the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19.

[0017] Embodiment 14: The bispecific antibody according to Embodiment 13, wherein VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 5 and 6 respectively; or wherein VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 14 and 15 respectively; or (3) the amino acid sequences shown by SEQ ID NO: 93 and 94 respectively.

[0018] Embodiment 15: The bispecific antibody according to Embodiment 13, wherein VL1, VH1, VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 1, 2, 10 and 11 respectively; (2) the amino acid sequences shown by SEQ ID NO: 1, 2, 14 and 15 respectively; (3) the amino acid sequences shown by SEQ ID NO: 1, 2, 93 and 94 respectively; (4) the amino acid sequences shown by SEQ ID NO: 5, 6, 10 and 11 respectively; or (5) the amino acid sequences shown by SEQ ID NO: 5, 6, 14 and 15 respectively; (6) the amino acid sequences shown by SEQ ID NO: 5, 6, 93 and 94 respectively.

[0019] Embodiment 16: The bispecific antibody according to Embodiment 13, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 3 and 30 respectively; (2) SEQ ID NO: 3 and 31 respectively; (3) SEQ ID NO: 7 and 32 respectively; (4) SEQ ID NO: 7 and 33 respectively; or (5) SEQ ID NO: 7 and 92 respectively.

[0020] Embodiment 17: The bispecific antibody according to Embodiment 11 or 12, wherein the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα.

[0021] Embodiment 18: The bispecific antibody according to Embodiment 17, wherein VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 14 and 15 respectively; or wherein VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 5 and 6 respectively.

[0022] Embodiment 19: The bispecific antibody according to Embodiment 17, wherein VL1, VH1, VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 10, 11, 1 and 2 respectively; (2) the amino acid sequences shown by SEQ ID NO: 10, 11, 5 and 6 respectively; (3) the amino acid sequences shown by SEQ ID NO: 14, 15, 1 and 2 respectively; or (4) the amino acid sequences shown by SEQ ID NO: 14, 15, 5 and 6 respectively.

[0023] Embodiment 20: The bispecific antibody according to Embodiment 17, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with (1) the amino acid sequences shown by SEQ ID NO: 12 and 34 respectively; (2) the amino acid sequences shown by SEQ ID NO: 12 and 35 respectively; (3) the amino acid sequences shown by SEQ ID NO: 16 and 36 respectively; or (4) the amino acid sequences shown by SEQ ID NO: 16 and 37 respectively.

[0024] Embodiment 21: A bispecific antibody that specifically binds to human TNFα and binds to human IL23p19, comprising: (1) a light chain (LC) that comprises a first light chain variable domain (VL1), a second light chain variable domain (VL2) and a CL region; and (2) a heavy chain (HC) that comprises a first heavy chain variable domain (VH1), a second heavy chain variable domain (VH2) and a CH region; wherein the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19 respectively, or specifically bind to human IL23p19 and human TNFα respectively.

[0025] Embodiment 22: The bispecific antibody according to Embodiment 21, wherein (1) the CL region is Cκ (SEQ ID NO: 68) or Cλ (SEQ ID NO: 69); or (2) the CH region is a human IgG1 CH region (SEQ ID NO: 58); or both (1) and (2).

[0026] Embodiment 23: The bispecific antibody according to Embodiment 21 or 22, wherein the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19.

[0027] Embodiment 24: The bispecific antibody according to Embodiment 23, wherein VL1 and VH1 have (1) amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) amino acid sequences shown by SEQ ID NO: 5 and 6 respectively; or wherein VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) amino acid sequences shown by SEQ ID NO: 14 and 15 respectively.

[0028] Embodiment 25: The bispecific antibody according to Embodiment 23, wherein VL1, VH1, VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 1, 2, 10 and 11 respectively; (2) amino acid sequences shown by SEQ ID NO: 1, 2, 14 and 15 respectively; (3) amino acid sequences shown by SEQ ID NO: 5, 6, 10 and 11 respectively; or (4) amino acid sequences shown by SEQ ID NO: 5, 6, 14 and 15 respectively.

[0029] Embodiment 26: The bispecific antibody according to Embodiment 23, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with (1) amino acid sequences shown by SEQ ID NO: 46 and 47 respectively; (2) amino acid sequences shown by SEQ ID NO: 48 and 49 respectively; (3) amino acid sequences shown by SEQ ID NO: 50 and 51 respectively; or (4) amino acid sequences shown by SEQ ID NO: 52 and 53 respectively.

[0030] Embodiment 27: The bispecific antibody according to Embodiment 21 or 22, wherein the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα.

[0031] Embodiment 28: The bispecific antibody according to Embodiment 27, wherein VL1 and VH1 have (1) amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) amino acid sequences shown by SEQ ID NO: 14 and 15 respectively; or wherein VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) amino acid sequences shown by SEQ ID NO: 5 and 6 respectively.

[0032] Embodiment 29: The bispecific antibody according to Embodiment 27, wherein VL1, VH1, VL2, and VH2 have (1) amino acid sequences represented by SEQ ID NO: 10, 11, 1, and 2, respectively; (2) amino acid sequences represented by SEQ ID NO: 10, 11, 5, and 6, respectively; (3) amino acid sequences represented by SEQ ID NO: 14, 15, 1, and 2, respectively, or (4) amino acid sequences represented by SEQ ID NO: 14, 15, 5, and 6, respectively.

[0033] Embodiment 30: The bispecific antibody according to Embodiment 27, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with (1) amino acid sequences represented by SEQ ID NO: 38 and 39, respectively; (1) amino acid sequences represented by SEQ ID NO: 40 and 41, respectively; (3) amino acid sequences represented by SEQ ID NO: 42 and 43, respectively; or (4) amino acid sequences represented by SEQ ID NO: 44 and 45, respectively.

[0034] Embodiment 31: A bispecific antibody that specifically binds to human TNFα and binds to human IL23p19, comprising (1) a light chain (LC) that comprises a light chain variable domain (VL) and a CL region; and (2) a heavy chain (HC) that comprises a heavy chain variable domain (VH) and a CH region; wherein (1) the VL / VH pair specifically binds to human IL23p19; and (2) the HC further comprises a single domain antibody of heavy chain variable domain (VHH) that specifically binds to human TNFα.

[0035] Embodiment 32: The bispecific antibody according to Embodiment 31, wherein (1) the CL region is Cκ (SEQ ID NO: 68) or Cλ (SEQ ID NO: 69); or (2) the CH region is a human IgG1 CH region (SEQ ID NO: 58); or both (1) and (2).

[0036] Embodiment 33: The bispecific antibody according to Embodiment 31 or 32, wherein VL and VH have (1) amino acid sequences represented by SEQ ID NO: 10 and 11, respectively; or (2) amino acid sequences represented by SEQ ID NO: 14 and 15, respectively.

[0037] Embodiment 34: The bispecific antibody according to any one of Embodiments 31 to 33, wherein VHH has the amino acid sequence represented by SEQ ID NO: 9.

[0038] Embodiment 35: The bispecific antibody according to any one of Embodiments 31 to 34, wherein VHH is linked to the N-terminus of VH.

[0039] Embodiment 36: The bispecific antibody according to Embodiment 35, wherein the LC and HC respectively have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 12 and 54 respectively; or (2) SEQ ID NO: 16 and 56 respectively.

[0040] Embodiment 37: The bispecific antibody according to any one of Embodiments 31 to 34, wherein the VHH is linked to the C-terminus of the Fc domain.

[0041] Embodiment 38: The bispecific antibody according to Embodiment 37, wherein the LC and HC respectively have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 12 and 55 respectively; or (2) SEQ ID NO: 16 and 57 respectively.

[0042] Embodiment 39: A pharmaceutical composition comprising the bispecific antibody according to any one of Embodiments 1 to 38 and a pharmaceutically acceptable carrier.

[0043] Embodiment 40: A method for reducing TNFα and / or IL23p19-related autoimmunity or inflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific antibody according to any one of Embodiments 1 to 38.

[0044] Embodiment 41: The method according to Embodiment 45, wherein the subject has an autoimmune disease or an inflammatory disease.

[0045] Embodiment 42: A method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific antibody according to any one of Embodiments 1 to 38.

[0046] Embodiment 43: A method for treating an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific antibody according to any one of Embodiments 1 to 38.

[0047] Embodiment 44: The method according to any one of Embodiments 45 to 48, wherein the subject is a human.

[0048] Embodiment 45: Use of the bispecific antibody according to any one of Embodiments 1 to 38 as a medicament.

[0049] Embodiment 46: Use of the bispecific antibody according to any one of Embodiments 1 to 38 in the treatment of autoimmune diseases.

[0050] Embodiment 47: Use of the bispecific antibody according to any one of Embodiments 1 to 38 in the treatment of inflammatory diseases.

[0051] Embodiment 48: Use of the bispecific antibody according to any one of Embodiments 1 to 38 for the preparation of a medicament for the treatment of autoimmune diseases.

[0052] Embodiment 49: Use of the bispecific antibody according to any one of Embodiments 1 to 38 for the preparation of a medicament for the treatment of inflammatory diseases.

[0053] Embodiment 50: A polynucleotide encoding LC1, LC2, HC1, HC2 or any combination thereof of the bispecific antibody according to any one of Embodiments 1 to 10.

[0054] Embodiment 51: A polynucleotide encoding LC, HC or both LC and HC of the bispecific antibody according to any one of Embodiments 11 to 38.

[0055] Embodiment 52: A vector comprising the polynucleotide according to Embodiment 50 or 51.

[0056] Embodiment 53: A cell comprising (a) the polynucleotide according to Embodiment 50 encoding LC1, LC2, HC1 and HC2, or (b) a plurality of polynucleotides according to Embodiment 50 co-encoding LC1, LC2, HC1 and HC2.

[0057] Embodiment 54: A cell comprising (a) the polynucleotide according to Embodiment 51 encoding both LC and HC, or (b) a first polynucleotide according to Embodiment 51 encoding LC and a second polynucleotide according to Embodiment 51 encoding HC.

[0058] Embodiment 55: A method for producing a bispecific antibody that specifically binds to human TNFα and binds to human IL23p19 by expressing the polynucleotide or the plurality of polynucleotides in the cell according to Embodiment 53 or 54. Description of the Drawings

[0059] Figures 1A - 1D Figures are provided showing 4 different types of anti-TNFa / IL23p19 bispecific antibodies disclosed herein. (N) represents the N-terminus; (C) represents the C-terminus.

[0060] Figure 1AShows the "CrossMab-KIH" structure, which includes four different polypeptides: a first light chain (LC1), a first heavy chain (HC1), a second light chain (LC2), and a second heavy chain (HC2), and its configuration is as shown below: LC1: (N)-VL1-CH1-(C) HC1: (N)-VH1-CL region-Fc region (stalk)-(C) LC2: (N)-VL2-CL region-(C) HC2: (N)-VH2-CH1-Fc region (socket)-(C)

[0061] Figure 1B Shows the "IgG-ScFv" structure, which includes the same two pairs of light chains (LC) and heavy chains (HC), and its configuration is as follows: LC: (N)-VL1-CL region-(C) HC: (N)-VH1-CH region-VH2-VL2-(C)

[0062] Figure 1C Shows the "DVD-Ig" structure, which includes the same two pairs of light chains (LC) and heavy chains (HC), and its configuration is as follows: LC: (N)-VL1-VL2-CL region-(C) HC: (N)-VH1-VH2-CH region-(C)

[0063] Figure 1D Shows the "SMAB-VHH" structure, which includes the same two pairs of light chains (LC) and heavy chains (HC), and its configuration is as follows: LC: (N)-VL1-CL region-(C) HC: (N)-VHH-VH-CH region-(C) or (N)-VH-CH region-VHH-(C)

[0064] Figure 2 Shows a graph summarizing the binding affinities of exemplary anti-TNFα / IL23p19 bispecific antibodies for TNFα and IL23p19, respectively.

[0065] Figure 3 Provides a graph showing the simultaneous binding of an exemplary anti-TNFα / IL23p19 bispecific antibody to two antigens, namely human TNFα and IL23p19.

[0066] Figures 4A - 4C Provides a graph showing the effective inhibition of TNFα-induced NFκB signaling by an exemplary anti-TNFα / IL23p19 bispecific antibody.

[0067] Figures 5A - 5C Figures showing effective inhibition of IL-23-induced STAT3 phosphorylation by an exemplary anti-TNFα / IL23p19 bispecific antibody.

[0068] Figure 6 Figures showing effective inhibition of TNFα-induced apoptosis of U937 cells by an exemplary anti-TNFα / IL23p19 bispecific antibody.

[0069] Figure 7 Figures showing effective inhibition of TNFα-induced cytotoxicity of L929 cells by an exemplary anti-TNFα / IL23p19 bispecific antibody.

[0070] Figure 8 Figures showing effective inhibition of the combination of TNFα with IL-23-induced human IL-17 cytokine production in human PBMCs by an exemplary anti-TNFα / IL23p19 bispecific antibody.

[0071] Figure 9 Figures showing effective inhibition of human TNFα-induced mIL-6 production in mice by an exemplary anti-TNFα / IL23p19 bispecific antibody.

[0072] Figure 10 Figures showing effective inhibition of human IL-23-induced ear hyperplasia in mice by an exemplary anti-TNFα / IL23p19 bispecific antibody, wherein ear thickness, clinical PASI score, and ear histopathology score were evaluated. DETAILED DESCRIPTION OF THE INVENTION

[0073] The present disclosure provides novel bispecific antibodies that specifically bind to human TNFα (TNFα) and to the p19 subunit of human IL-23 (IL23p19). Also disclosed herein are pharmaceutical compositions comprising a therapeutically effective amount of these antibodies. Also disclosed herein are the uses of these antibodies and pharmaceutical compositions for reducing autoimmunity and for treating autoimmune and inflammatory diseases.

[0074] Prior to further describing the present disclosure, it is to be understood that the present disclosure is not limited to the specific embodiments described herein and that the terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting. 1. Definitions

[0075] Unless otherwise defined herein, scientific and technical terms used in this disclosure of the invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Generally, the terms used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, as well as the techniques of these fields, are those well known and commonly used in the art.

[0076] The term "a" entity refers to one or more of that entity; for example, "an antibody" is understood to represent one or more antibodies.

[0077] As used herein, the term "and / or" will be regarded as a specific disclosure of each of the two specified features or components with or without the other. Thus, herein, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", (alone) "A"; and (alone) "B". Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; (alone) A; (alone) B; and (alone) C.

[0078] The terms "polypeptide", "peptide", "protein", "polypeptide chain", "peptide chain" and their grammatical equivalents, as used interchangeably herein, refer to polymers of amino acids of any length, which may be linear or branched. It may include non-natural or modified amino acids or may be interrupted by non-amino acids. The polypeptide, peptide, polypeptide chain, peptide chain or protein may also be modified by (e.g.) disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other operation or modification.

[0079] The terms "polynucleotide", "nucleic acid" and their grammatical equivalents, as used interchangeably herein, denote polymers of nucleotides of any length and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be introduced into the polymer by DNA or RNA polymerase.

[0080] As used herein, the term "variant" with respect to a protein or polypeptide having specific sequence characteristics ("reference protein" or "reference polypeptide") refers to a distinct protein or polypeptide that has one or more (such as (e.g.) from about 1 to about 25, from about 1 to about 20, from about 1 to about 15, from about 1 to about 10, or from about 1 to about 5) amino acid substitutions, deletions, and / or additions as compared to the reference protein or polypeptide. The changes in the amino acid sequence can be amino acid substitutions. The changes in the amino acid sequence can be conservative amino acid substitutions. Functional fragments or functional variants of a protein or polypeptide maintain the basic structural and functional properties of the reference protein or polypeptide.

[0081] As used herein, the term "specifically binds" means that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or in some combination of the foregoing, as compared to alternative substances, including related and unrelated proteins. For example, a binding moiety (e.g., an antibody) that specifically binds a target molecule (e.g., an antigen) can be identified by immunoassays, ELISA, biolayer interferometry ("BLI"), SPR (e.g., Biacore), or other techniques known to those of skill in the art. Typically, a specific reaction will be at least two-fold the background signal or noise and can be greater than 10-fold the background. For a discussion of antibody specificity, see, e.g., Paul, ed., 1989, FUNDAMENTAL IMMUNOLOGY SECOND EDITION, Raven Press, New York, pp. 332-336. A binding moiety that specifically binds a target molecule can bind the target molecule with a higher affinity than its affinity for different molecules. In some embodiments, a binding moiety that specifically binds a target molecule can bind the target molecule with an affinity that is at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, or at least 100-fold greater than its affinity for different molecules. In some embodiments, a binding moiety that specifically binds a particular target molecule binds different molecules with a low affinity such that binding is undetectable using assays described herein or otherwise known in the art. In some embodiments, "specifically binds" means (e.g.) that the binding moiety binds with a K of about 0.1 mM or less D to a molecular target. In some embodiments, "specifically binds" means that a polypeptide or molecule binds with a K of about 10 M or less or about 1 μM or less D to a target. In some embodiments, "specifically binds" refers to a polypeptide or molecule that binds with a K of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less DBinding target. Due to the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It should be understood that in some embodiments, a binding moiety (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (although it may include) exclusive binding, i.e., binding to a single target. Accordingly, in some embodiments, a binding moiety (e.g., an antibody) can specifically bind to more than one target. For example, in certain cases, an antibody can contain two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, the antibody can be bispecific and contain at least two antigen-binding sites with different specificities.

[0082] As used herein, the term "binding affinity" generally refers to the total strength of the non-covalent interactions between a binding moiety and a target molecule (e.g., an antigen). The binding between a binding moiety and a target molecule is a reversible process, and the binding affinity is typically reported as the equilibrium dissociation constant (K D ). K D is the ratio of the dissociation rate (k off or K d ) to the association rate (k on or k a ). The lower the K D of a binding pair, the higher the affinity. A variety of methods for measuring binding affinity are known in the art, and any of them can be used for the purposes disclosed in the present invention. Specific illustrative embodiments include the following. In some embodiments, "K D " or "K D value" can be measured by assays known in the art, e.g., by a binding assay. K D can be measured in a radioimmunoassay (RIA) of radiolabeled antigen binding (Chen et al., (1999) J. Mol Biol 293:865-881). K D or K D value can also be measured using biolayer interferometry (BLI) by using (e.g.) the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). K Dor K D value

[0083] As used herein in the context of two or more polynucleotides or polypeptides, the terms "identical," "percent identity," and their grammatical equivalents denote two or more sequences or subsequences that have the same or a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary) and without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software for obtaining amino acid or nucleotide sequence alignments are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, the GCG Wisconsin Package and variations thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, which means that when compared and aligned for maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleotide or amino acid residue identity, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists within an amino acid sequence region of at least about 10 residues, at least about 20 residues, at least about 40 - 60 residues, at least about 60 - 860 residues or any integer value therebetween. In some embodiments, identity exists within a region of greater than 60 - 80 residues, such as at least about 80 - 100 residues, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared (such as the coding region of a target protein or antibody). In some embodiments, identity exists within a nucleotide sequence region of at least about 10 bases, at least about 20 bases, at least about 40 - 60 bases, at least about 60 - 80 bases or any integer value therebetween. In some embodiments, identity exists within a region of greater than 60 - 80 bases, such as at least about 80 - 1000 bases or more bases, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared (such as the nucleotide sequence encoding a protein of interest).

[0084] A "separated" polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition in a form that does not exist in nature. Separated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to the extent that they are no longer in the form in which they exist in nature. In some embodiments, the separated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells or compositions are substantially pure.

[0085] Ranges: Throughout the disclosure of the invention, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within the range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values within the range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0086] Exemplary genes and polypeptides are described herein with reference to GenBank accession numbers, GI numbers, and / or SEQ ID NOs. It should be understood that those skilled in the art can readily identify homologous sequences by referring to sequence sources, including (but not limited to) Uniprot (https: / / www.uniprot.org / ), GenBank (ncbi.nlm.nih.gov / genbank / ), and EMBL (embl.org / ). 2. Bispecific antibody targeting TNFα and IL23p19

[0087] Bispecific antibodies that specifically bind to both human TNFα and human IL23p19 are provided herein. In some embodiments, the bispecific antibodies provided herein are monoclonal antibodies. In some embodiments, the bispecific antibodies provided herein are separated. In some embodiments, the bispecific antibodies provided herein are substantially pure. 2.1 Conventional

[0088] Tumor necrosis factor α (TNFα) is a pleiotropic homotrimeric cytokine. TNFα is mainly secreted by monocytes, macrophages, lymphocytes, endothelial cells, and fibroblasts. TNFα binds to two different receptors: TNFRI, which is expressed on almost all cell types, and TNFRII, which is expressed limitedly on immune cells (CD4+ T cells, NK cells). TNFα is expressed in both a soluble form and a transmembrane form (the membrane-bound precursor form can be proteolytically cleaved by the metalloproteinase TNFα converting enzyme (TACE) into a soluble homotrimer). Both the membrane-bound form and the soluble form of the cytokine are biologically active. TNFα is a potent inducer of the inflammatory response. It promotes the production of pro-inflammatory cytokines and chemokines, enhances the recruitment and infiltration of leukocytes, and activates both innate and adaptive immunity. As such, TNFα can be important in systemic inflammation, particularly the acute-phase inflammatory response. Excessive TNFα has been associated with various forms of autoimmune diseases. Silva et al., Immunotherapy (2010) 2(6), 817-833; Salomon, Nat. Rev. Rheumatol. 17(8):487-504 (2021). The following provides an exemplary amino acid sequence of human TNFα: MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQR EEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELR DNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRE TPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO:74; Uniprot ID: P01375-1)

[0089] Interleukin-23 (IL23) is a heterodimeric cytokine composed of two subunits, p40 (shared with IL12) and p19 (unique to IL23). The p19 subunit is also known as IL23A. IL23 binds to a cell surface receptor composed of the IL12 receptor β1 subunit and the unique IL23 receptor subunit. Expression of IL23R is restricted to specific immune cell populations and is predominantly present on T cells (αβ and γδ TCR+) and NK cell subsets. IL23 is an upstream regulator of IL-6, IL-17, GM-CSF, and IL-22 in tissue inflammation. IL23 signals downstream via TYK2 / JAK2-mediated phosphorylation of STAT3. IL23 promotes the development and differentiation of naive CD4+ T cells into pathogenic Th17 cells and stimulates iNKT cells, γδ T cells, and ILC3 to produce IL17 family cytokines. IL23 also promotes osteoclastogenesis and bone resorption. IL23 promotes the activation of a range of inflammatory cells involved in chronic inflammation induction, regulating both memory / pathogenic T cell inflammatory responses and innate lymphoid cell inflammatory activity. As such, elevated IL23 production has been implicated as a major factor in inflammatory and autoimmune diseases. Moschen et al., Nat. Rev. Gastroenterol. Hepatol. 16(3):185-196 (2019); Schmitt et al., Front Immunol. 2021; 12:622934; Silvagni et al., Front Pharmacol., 2021; 12:672515. The following provides an exemplary amino acid sequence of the p19 subunit of human IL23: MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEG DEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSP VGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVF AHGAATLSP (SEQ ID NO:75; Uniprot ID: Q9NPF7)

[0090] As used herein and as understood in the art, an "antibody" is an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof, through at least one antigen-binding site that is typically located within the variable region of the immunoglobulin molecule. A "bispecific" antibody is an artificial hybrid antibody that has two different antigen-binding sites and recognizes and specifically binds to two different target antigens.

[0091] The term "antibody" is used herein in its broadest sense to encompass antibodies of different types and structures, including polyclonal antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule that contains an antigen-binding site (e.g., a dual variable domain immunoglobulin molecule), provided that the antibody exhibits the desired biological activity. Antibodies also include (but are not limited to) murine antibodies, camelid antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Based on the properties of their heavy chain constant domains, which are designated α, δ, ε, γ, and μ, respectively, antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Unless otherwise expressly indicated, the term "antibody" as used herein includes "antigen-binding fragments" of intact antibodies. The term "antigen-binding fragment" as used herein refers to a portion or fragment of an intact antibody that is the antigen-determining variable region of the intact antibody. Examples of antigen-binding fragments include (but are not limited to) Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy chain antibodies (HCAb), light chain antibodies (LCAb), disulfide-linked scFv (dsscFv), diabodies, triabodies, tetra-bodies, microantibodies, dual variable domain antibodies (DVD), single variable domain antibodies (sdAb; e.g., camelid antibodies, alpaca antibodies), and single variable domains of heavy chain antibodies (VHH), as well as bispecific or multispecific antibodies formed from antigen fragments.

[0092] The structure of immunoglobulins has been well characterized (see, e.g., Chapter 7 of FUNDAMENTAL IMMUNOLOGY (Paul, W. ed., 2nd ed. Raven Press, N.Y. (1989))). Generally, an immunoglobulin contains two pairs of polypeptide chains, one pair of light chains (L; low molecular weight) and one pair of heavy chains (H; high molecular weight), and all four chains are interconnected by disulfide bonds.

[0093] Each light chain of an immunoglobulin generally includes a variable light chain region (“VL region”) and a constant light chain region (“CL region”). There are two different types of light chains, which are designated as kappa (κ) or lambda (λ) based on the amino acid sequence of the CL region. The amino acid sequences of the CL regions are well known in the art.

[0094] Each heavy chain generally includes a variable heavy chain region (“VH region”) and a constant heavy chain region (“CH region”). The VH region can be one of five different types, which are designated as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence. When combined with a light chain, these different types of heavy chains give rise to five well-known antibody classes, namely IgA, IgD, IgE, IgG, and IgM. There are four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4. The amino acid sequences of the CH regions of the different antibody classes are well known in the art.

[0095] The CH region of an immunoglobulin contains more than one domain. For example, the CH region of an IgG antibody consists of three domains, the constant heavy domain 1 (CH1), the constant heavy domain 2 (CH2), and the constant heavy domain 3 (CH3). The highly flexible region between the CH1 and CH2 domains is referred to as the “hinge region”. The disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an immunoglobulin. The “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. In the IgG, IgA, and IgD isotypes, the Fc region consists of the CH2 domain and the CH3 domain; the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4). The amino acid sequences of the Fc regions of human IgG, IgA, IgD, IgM, and IgE and the subtypes IgG1, IgG2, IgG3, and IgG4 are known to those skilled in the art. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from the hinge region to the carboxyl terminus of the heavy chain.

[0096] As used herein, the term “Fc region” includes native sequence Fc regions and variant Fc regions. In some embodiments, the Fc domains of the two heavy chains of the bispecific antibodies provided herein may contain paired modifications that promote their binding to each other rather than forming homodimers.

[0097] Unless otherwise specified or inconsistent with the context, the amino acid positions in the constant regions are numbered according to EU-numbering (Edelman et al., PNAS. 1969; 63:78-85, Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed. 1991 NIH Publication No. 91-3242). The following provides a list of exemplary amino acid sequences of the constant domains / regions of human IgG antibodies as Table 1.

[0098] Table 1. Amino acid sequences of human IgG constant regions / domains.

[0099] The term "variable region" refers to the part of the light or heavy chain of an immunoglobulin that is generally located at the amino terminus of the light or heavy chain and is used in the binding and specificity of each particular antibody for its specific antigen. The light chain variable region is referred to as the "light chain variable region" or "VL region", which includes at least one, usually one "light chain variable domain" or "VL". The heavy chain variable region is referred to as the "heavy chain variable region" or "VH region", which includes at least one, usually one "heavy chain variable domain" or "VH". The sequences of the variable domains vary widely between different antibodies. The VL and VH pairs can bind and form a binding site that specifically binds to a target antigen or epitope.

[0100] The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions that can be hypervariable in the sequence and / or form of structurally defined loops), also known as complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The sequence variations are concentrated in the CDRs, and the less variable parts of the variable domain are called framework regions (FRs). The CDRs of the light and heavy chains are mainly responsible for the interaction of the antibody with the antigen. Each VH and VL usually consists of 3 CDRs and 4 FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, J Mol Biol 1987; 196:901-17).

[0101] CDR refers to one of three hypervariable regions (H1, H2, or H3) within the non-framework regions of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of three hypervariable regions (L1, L2, or L3) within the non-framework regions of the VL β-sheet framework of an antibody. CDR regions are well known to those skilled in the art and have been defined by multiple methods / systems. These systems and / or definitions have been developed and refined over several years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defined the most variable regions within the antibody variable (V) domain (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). Software programs (e.g., abYsis) are available and known to those skilled in the art for analyzing antibody sequences and determining CDRs.

[0102] Disclosed herein are bispecific antibodies having different structures, which include VH and VL that specifically bind to human TNFα or human IL23p19. Although specific anti-human TNFα and anti-human IL23p19 VH / VLs are illustrated herein, those of ordinary skill in the art will understand that the bispecific antibodies disclosed herein are not limited to the illustrated VH / VLs. Also specifically contemplated herein are variants of the bispecific antibodies disclosed herein, wherein the illustrated anti-human TNFα and anti-human IL23p19 VH / VLs are replaced with other anti-human TNFα and anti-human IL23p19 VH / VLs. Lists of exemplary anti-human TNFα or anti-human IL23p19 VH / VLs are provided below as Table 2A and Table 2B, respectively.

[0103] Table 2A: Amino acid sequences of VH / VLs of exemplary anti-human TNFα antibodies

[0104] Table 2B: Amino acid sequences of VH / VLs of exemplary anti-human IL23p19 antibodies 2.2 CrossMab-KIH

[0105] In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and bind to human IL23p19 have Figure 1A the "CrossMab-KIH" structure shown. The "knobs-into-holes" or "KIH" model facilitates the formation of heterodimers of engineered bispecific antibodies rather than homodimers of heavy chains.

[0106] Modifications that promote the association of Fc domain pairs in bispecific antibodies include so-called "knob-into-hole" modifications, which include a "knob" modification in one Fc domain and a "hole" modification in the other. The knob-into-hole technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot. Eng. 9, 617-621 (1996) and Carter, J. Immunol. Meth. 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of the first Fc ("knob-Fc") and a corresponding cavity ("hole") at the interface of the second Fc ("hole-Fc"), such that the protrusion can be positioned in the cavity to promote heterodimer formation and impede homodimer formation. The protrusion is constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0107] Thus, "knob-Fc regions" and "hole-Fc regions" are designed to form heterodimer pairs. A knob-Fc region refers to a region in which the amino acids of the CH3 domain are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion within the CH3 domain that can be positioned within a cavity in the CH3 domain of the hole-Fc region, in which the amino acid residues of the CH3 domain are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity within the CH3 domain within which the protrusion within the CH3 domain of the first subunit is locatable. Preferably, the amino acid residues having a larger side chain volume are selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, e.g., by site-specific mutagenesis or by peptide synthesis.

[0108] In some embodiments, the threonine residue at position 366 of the pestle-Fc region is replaced with a tryptophan residue (T366W) and the tyrosine residue at position 407 of the mortar-Fc region is replaced with a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A). In some embodiments, the pestle-Fc region further has a serine residue at position 354 replaced with a cysteine residue (S354C) or a glutamate residue at position 356 replaced with a cysteine residue (E356C), and the mortar-Fc region further has a tyrosine residue at position 349 replaced with a cysteine residue (Y349C). In some embodiments, the pestle-Fc region contains the amino acid substitutions S354C and T366W, and the mortar-Fc region contains the amino acid substitutions Y349C, T366S, L368A and Y407V. All amino acid residues are numbered according to the EU index.

[0109] The CrossMab is designed to address BsAb light chain mispairing by swapping one side of CL and CH1. By swapping one side of the heavy and light chain domains, the BsAb light chains can be correctly assembled.

[0110] Thus, in some embodiments, the bispecific antibodies provided herein can have two pairs of light and heavy chains. In some embodiments, the first light and heavy chain pair (LC1 and HC1) specifically binds to human TNFα and the second light and heavy chain pair (LC2 and HC2) specifically binds to human IL23p19. In some embodiments, the LC1 and HC1 pair specifically binds to human IL23p19 and the LC2 and HC2 pair specifically binds to human TNFα. The HC1 and HC2 pairs of the bispecific antibody can employ the KIH design, wherein HC1 includes the pestle-Fc region and HC2 includes the mortar-Fc region. Alternatively, in some embodiments, HC2 can include the pestle-Fc region and HC1 can include the mortar-Fc region. Additionally, the light chain constant region (CL region) of LC1 and the heavy chain constant domain 1 (CH1) of HC1 are exchanged to avoid light chain mispairing. Thus, the bispecific antibodies provided herein can have LC1, HC1, LC2 and HC2, wherein (1) the LC1 / HC1 pair has exchanged constant domains, (2) the LC2 / HC2 pair has normal constant domains; and (3) HC1 / HC2 can have the KIH structure. In some embodiments, HC1 has the pestle-Fc region and HC2 has the mortar-Fc region. In some embodiments, HC1 has the mortar-Fc region and HC2 has the pestle-Fc region.

[0111] In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and bind to human IL23p19 can have four polypeptides, including: (1) a first light chain (LC1) that includes a first light chain variable domain (VL1) and a first heavy chain constant domain 1 (CH1); (2) a first heavy chain (HC1) that includes a first heavy chain variable domain (VH1), a first light chain constant region (CL), and a stamen-Fc region; (3) a second light chain (LC2) that includes a second light chain variable domain (VL2) and a second CL region; and (4) a second heavy chain (HC2) that includes a second heavy chain variable domain (VH2), a second CH1 domain, and a pistil-Fc region; wherein (i) the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively; and (ii) the stamen-Fc region is a variant of the human Fc region with a T366W substitution (according to EU index numbering); and the pistil-Fc region is a variant of the human Fc region with a Y407V substitution (according to EU index numbering).

[0112] The amino acid sequences of the CH1, CL regions, and Fc regions (CH2 and CH3) of the bispecific antibodies disclosed herein can be derived from any suitable source, e.g., the constant regions of antibodies such as IgG1, IgG2, IgG3, or IgG4. The amino acid sequences of the heavy and light chain constant regions of antibodies are well known in the art, e.g., those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0113] In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG1. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG2. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG3. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG4. In some embodiments, the amino acid sequences of the CH1, CL regions, and Fc regions (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein can include one or more amino acid substitutions that are different from the wild-type immunoglobulin, e.g., one or more amino acid substitutions in wild-type IgG1 or IgG4. These substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US 8323962, US6737056, and US7416727).

[0114] In some embodiments, the first and second CH1 domains can be independently selected from the human IgG1 CH1 domain (SEQ ID NO:61), the human IgG2 CH1 domain (SEQ ID NO:83), the human IgG3 CH1 domain (SEQ ID NO:84), and the human IgG4 CH1 domain (SEQ ID NO:85). In some embodiments, both the first and second CH1 domains are the human IgG1 CH1 domain (SEQ ID NO:61).

[0115] In some embodiments, the CL region can be κCL (Cκ; SEQ ID NO:68). In some embodiments, the CL region can be λCL (Cλ, SEQ ID NO:69). In some embodiments, the first CL region is Cκ (SEQ ID NO:68). In some embodiments, the first CL region is Cλ (SEQ ID NO:69). In some embodiments, the second CL region is Cκ (SEQ ID NO:68). In some embodiments, the second CL is Cλ (SEQ ID NO:69). In some embodiments, the first CL region is Cλ (SEQ ID NO:69) and the second CL region is Cκ (SEQ ID NO:68). In some embodiments, the first CL region is Cλ (SEQ ID NO:69) and the second CL region is Cλ (SEQ ID NO:69). In some embodiments, the first CL region is Cκ (SEQ ID NO:68) and the second CL region is Cκ (SEQ ID NO:68). In some embodiments, the first CL region is Cκ (SEQ ID NO:68) and the second CL region is Cλ (SEQ ID NO:69).

[0116] In some embodiments, the hinge of the Fc region can be independently selected from the human IgG1 hinge region (SEQ ID NO:70), the human IgG2 hinge region (SEQ ID NO:89), the human IgG3 hinge region (SEQ ID NO:90), and the human IgG4 hinge region (SEQ ID NO:91).

[0117] In some embodiments, the Fc region of the bispecific antibodies provided herein can be a variant of the Fc region of human IgG1. In some embodiments, the pestle-Fc region is human IgG1 Fc with a T366W substitution. In some embodiments, the mortar-Fc region is human IgG1 Fc with a Y407T substitution. In some embodiments, the pestle-Fc and mortar-Fc regions can further include S354C and Y349C substitutions, respectively. In some embodiments, the pestle-Fc and mortar-Fc regions can further include E356C and Y349C substitutions, respectively. In some embodiments, the mortar-Fc region can further include T366S and L368A substitutions. All amino acid residues are numbered according to the EU index. In some embodiments, the pestle-Fc region can have the amino acid sequence shown in SEQ ID NO:66. In some embodiments, the mortar-Fc region can have the amino acid sequence shown in SEQ ID NO:67. The following provides a list of exemplary Fc sequences in the KIH model as Table 3.

[0118] Table 3: Exemplary Fc sequences in the KIH model.

[0119] In some embodiments of the bispecific antibodies provided herein, (1) the first CL region is kappa CL (Cκ; SEQ ID NO:68) or lambda CL (Cλ, SEQ ID NO:69); and the second CL region is Cκ (SEQ ID NO:68) or Cλ (SEQ ID NO:69); (2) both the first CH1 domain and the second CH1 domain are human IgG1 CH1 domains (SEQ ID NO:61); or (3) the pestle-Fc region has the amino acid sequence shown in SEQ ID NO:66; and the mortar-Fc region has the amino acid sequence shown in SEQ ID NO:67; or any combination of (1)-(3).

[0120] In some embodiments of the bispecific antibodies provided herein, the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19. The VL1 / VH1 pair can be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL1 and VH1 are the VL and VH of adalimumab and have the amino acid sequences shown by SEQ ID NO:1 and 2, respectively. In some embodiments, VL1 and VH1 are the VL and VH of golimumab and have the amino acid sequences shown by SEQ ID NO:5 and 6, respectively. The VL2 / VH2 pair can be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL2 and VH2 are the VL and VH of guselkumab and have the amino acid sequences shown by SEQ ID NO:10 and 11, respectively. In some embodiments, VL2 and VH2 are the VL and VH of tildrakizumab and have the amino acid sequences shown by SEQ ID NO:14 and 15, respectively.

[0121] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have the amino acid sequences shown by (1) SEQ ID NO:1 and 2, respectively; or (2) SEQ ID NO:5 and 6, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown by SEQ ID NO:1 and 2, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown by SEQ ID NO:5 and 6, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have the amino acid sequences shown by (1) SEQ ID NO:10 and 11, respectively; or (2) SEQ ID NO:14 and 15, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown by SEQ ID NO:10 and 11, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown by SEQ ID NO:14 and 15, respectively.

[0122] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have (1) amino acid sequences set forth by SEQ ID NO: 1, 2, 10, and 11, respectively; (2) amino acid sequences set forth by SEQ ID NO: 1, 2, 14, and 15, respectively; (3) amino acid sequences set forth by SEQ ID NO: 5, 6, 10, and 11, respectively; or (4) amino acid sequences set forth by SEQ ID NO: 5, 6, 14, and 15, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences set forth by SEQ ID NO: 1, 2, 10, and 11, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences set forth by SEQ ID NO: 1, 2, 14, and 15, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences set forth by SEQ ID NO: 5, 6, 10, and 11, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences set forth by SEQ ID NO: 5, 6, 14, and 15, respectively.

[0123] In some embodiments of the bispecific antibodies provided herein, the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα. The VL1 / VH1 pair can be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL1 and VH1 are the VL and VH of guselkumab and have amino acid sequences set forth by SEQ ID NO: 10 and 11, respectively. In some embodiments, VL1 and VH1 are the VL and VH of tildrakizumab and have amino acid sequences set forth by SEQ ID NO: 14 and 15, respectively. The VL2 / VH2 pair can be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL2 and VH2 are the VL and VH of adalimumab and have amino acid sequences set forth by SEQ ID NO: 1 and 2, respectively. In some embodiments, VL2 and VH2 are the VL and VH of golimumab and have amino acid sequences set forth by SEQ ID NO: 5 and 6, respectively.

[0124] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have (1) amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) amino acid sequences shown by SEQ ID NO: 14 and 15 respectively. In some embodiments, VL1 and VH1 have amino acid sequences shown by SEQ ID NO: 10 and 11 respectively. In some embodiments, VL1 and VH1 have amino acid sequences shown by SEQ ID NO: 14 and 15 respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) amino acid sequences shown by SEQ ID NO: 5 and 6 respectively. In some embodiments, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 1 and 2 respectively. In some embodiments, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 5 and 6 respectively.

[0125] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have (1) amino acid sequences shown by SEQ ID NO: 10, 11, 1, and 2 respectively; (2) amino acid sequences shown by SEQ ID NO: 10, 11, 5, and 6 respectively; (3) amino acid sequences shown by SEQ ID NO: 14, 15, 1, and 2 respectively; or (4) amino acid sequences shown by SEQ ID NO: 14, 15, 5, and 6 respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 10, 11, 1, and 2 respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 10, 11, 5, and 6 respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 14, 15, 1, and 2 respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 14, 15, 5, and 6 respectively. The following provides a list of exemplary VL1, VH1, VL2, and VH2 of bispecific antibodies as Table 4A.

[0126] Table 4A: Exemplary bispecific antibodies against human TNFα and human IL23p19

[0127] In some embodiments, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 18, 19, 3, and 20, respectively; (2) SEQ ID NO: 21, 22, 3, and 20, respectively; (3) SEQ ID NO: 18, 19, 7, and 23, respectively; (4) SEQ ID NO: 21, 22, 7, and 23, respectively; (5) SEQ ID NO: 24, 25, 12, and 26, respectively; (6) SEQ ID NO: 27, 28, 12, and 26, respectively; (7) SEQ ID NO: 24, 25, 16, and 29, respectively; or (8) SEQ ID NO: 27, 28, 16, and 29, respectively.

[0128] In some embodiments, provided herein are dual - specific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 3, and 20, respectively. In some embodiments, LC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18. In some embodiments, LC1 has the amino acid sequence shown by SEQ ID NO: 18. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, HC1 has the amino acid sequence shown by SEQ ID NO: 19. In some embodiments, LC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 3. In some embodiments, LC2 has the amino acid sequence shown by SEQ ID NO: 3. In some embodiments, HC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, HC2 has the amino acid sequence shown by SEQ ID NO: 20. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 3, and 20, respectively. In some embodiments, provided herein is a dual - specific antibody designated as A1, wherein LC1, HC1, LC2, and HC2 have the amino acid sequences shown by SEQ ID NO: 18, 19, 3, and 20, respectively.

[0129] In some embodiments, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences shown by SEQ ID NO: 21, 22, 3, and 20, respectively. In some embodiments, LC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21. In some embodiments, LC1 has the amino acid sequence shown by SEQ ID NO: 21. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22. In some embodiments, HC1 has the amino acid sequence shown by SEQ ID NO: 22. In some embodiments, LC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, LC2 has the amino acid sequence shown by SEQ ID NO: 3. In some embodiments, HC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, HC2 has the amino acid sequence shown by SEQ ID NO: 20. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO: 21, 22, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO: 21, 22, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO: 21, 22, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO: 21, 22, 3, and 20, respectively. In some embodiments, provided herein is a bispecific antibody designated as A2, wherein LC1, HC1, LC2, and HC2 have the amino acid sequences shown by SEQ ID NO: 21, 22, 3, and 20, respectively.

[0130] In some embodiments, provided herein are dual - specific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 7, and 23, respectively. In some embodiments, LC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18. In some embodiments, LC1 has the amino acid sequence shown by SEQ ID NO: 18. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, HC1 has the amino acid sequence shown by SEQ ID NO: 19. In some embodiments, LC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7. In some embodiments, LC2 has the amino acid sequence shown by SEQ ID NO: 7. In some embodiments, HC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23. In some embodiments, HC2 has the amino acid sequence shown by SEQ ID NO: 23. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 7, and 23, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 7, and 23, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 7, and 23, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 18, 19, 7, and 23, respectively. In some embodiments, provided herein is a dual - specific antibody designated as A3, wherein LC1, HC1, LC2, and HC2 have the amino acid sequences shown by SEQ ID NO: 18, 19, 7, and 23, respectively.

[0131] In some embodiments, provided herein are dual - specific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by SEQ ID NO:21, 22, 7, and 23, respectively. In some embodiments, LC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:21. In some embodiments, LC1 has the amino acid sequence shown by SEQ ID NO:21. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:22. In some embodiments, HC1 has the amino acid sequence shown by SEQ ID NO:22. In some embodiments, LC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:7. In some embodiments, LC2 has the amino acid sequence shown by SEQ ID NO:7. In some embodiments, HC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:23. In some embodiments, HC2 has the amino acid sequence shown by SEQ ID NO:23. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO:21, 22, 7, and 23, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO:21, 22, 7, and 23, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO:21, 22, 7, and 23, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO:21, 22, 7, and 23, respectively. In some embodiments, provided herein is a dual - specific antibody designated as A4, wherein LC1, HC1, LC2, and HC2 have the amino acid sequences shown by SEQ ID NO:21, 22, 7, and 23, respectively.

[0132] In some embodiments, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 12, and 26, respectively. In some embodiments, LC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 24. In some embodiments, LC1 has the amino acid sequence shown by SEQ ID NO: 24. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 25. In some embodiments, HC1 has the amino acid sequence shown by SEQ ID NO: 25. In some embodiments, LC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 12. In some embodiments, LC2 has the amino acid sequence shown by SEQ ID NO: 12. In some embodiments, HC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 26. In some embodiments, HC2 has the amino acid sequence shown by SEQ ID NO: 26. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 12, and 26, respectively. In some embodiments, provided herein is a bispecific antibody designated as A5, wherein LC1, HC1, LC2, and HC2 have the amino acid sequences shown by SEQ ID NO: 24, 25, 12, and 26, respectively.

[0133] In some embodiments, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences shown by SEQ ID NO: 27, 28, 12, and 26, respectively. In some embodiments, LC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27. In some embodiments, LC1 has the amino acid sequence shown by SEQ ID NO: 27. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28. In some embodiments, HC1 has the amino acid sequence shown by SEQ ID NO: 28. In some embodiments, LC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, LC2 has the amino acid sequence shown by SEQ ID NO: 12. In some embodiments, HC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26. In some embodiments, HC2 has the amino acid sequence shown by SEQ ID NO: 26. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO: 27, 28, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO: 27, 28, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO: 27, 28, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO: 27, 28, 12, and 26, respectively. In some embodiments, provided herein is a bispecific antibody designated A6, wherein LC1, HC1, LC2, and HC2 have the amino acid sequences shown by SEQ ID NO: 27, 28, 12, and 26, respectively.

[0134] In some embodiments, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 16, and 29, respectively. In some embodiments, LC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 24. In some embodiments, LC1 has the amino acid sequence shown by SEQ ID NO: 24. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 25. In some embodiments, HC1 has the amino acid sequence shown by SEQ ID NO: 25. In some embodiments, LC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 16. In some embodiments, LC2 has the amino acid sequence shown by SEQ ID NO: 16. In some embodiments, HC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29. In some embodiments, HC2 has the amino acid sequence shown by SEQ ID NO: 29. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 16, and 29, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 16, and 29, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 16, and 29, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 24, 25, 16, and 29, respectively. In some embodiments, provided herein is a bispecific antibody designated as A7, wherein LC1, HC1, LC2, and HC2 have the amino acid sequences shown by SEQ ID NO: 24, 25, 16, and 29, respectively.

[0135] In some embodiments, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19 and have LC1, HC1, LC2, and HC2, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 27, 28, 16, and 29, respectively. In some embodiments, LC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 27. In some embodiments, LC1 has the amino acid sequence shown by SEQ ID NO: 27. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28. In some embodiments, HC1 has the amino acid sequence shown by SEQ ID NO: 28. In some embodiments, LC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 16. In some embodiments, LC2 has the amino acid sequence shown by SEQ ID NO: 16. In some embodiments, HC2 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29. In some embodiments, HC2 has the amino acid sequence shown by SEQ ID NO: 29. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 27, 28, 16, and 29, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 27, 28, 16, and 29, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 27, 28, 16, and 29, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 27, 28, 16, and 29, respectively. In some embodiments, provided herein is a bispecific antibody designated as A8, wherein LC1, HC1, LC2, and HC2 have the amino acid sequences shown by SEQ ID NO: 27, 28, 16, and 29, respectively. 2.3IgG-ScFv

[0136] In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and bind to human IL23p19 have Figure 1B the "IgG-ScFv" structure shown in Figure 1B . As shown, the scFv comprising a VH / VL pair that specifically binds to a first antigen is linked to the CH3 domain of an IgG that specifically binds to a second antigen. In some embodiments, the N-terminus of the scFv is linked to the C-terminus of the heavy chain of the IgG. In some embodiments, the N-terminus of the scFv is linked to the C-terminus of the CH3 domain of the IgG. In some embodiments, the N-terminus of the scFv is linked to the C-terminus of the light chain of the IgG. In some embodiments, the N-terminus of the scFv is linked to the C-terminus of the VL region of the IgG. In some embodiments, the scFv has a linker that links VH and VL. In some embodiments of the scFv, the N-terminus of VH in the scFv is linked to the C-terminus of VL. In some embodiments of the scFv, the N-terminus of VL in the scFv is linked to the C-terminus of VH.

[0137] In some embodiments, the scFv specifically binds to human TNFα and the IgG specifically binds to human IL23p19. In some embodiments, the scFv specifically binds to human IL23p19 and the IgG specifically binds to human TNFα.

[0138] Accordingly, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19, comprising (1) a light chain (LC) comprising a first light chain variable domain (VL1) and a CL region; and (2) a heavy chain (HC) comprising a first heavy chain variable domain (VH1), a heavy chain constant region (CH), a second light chain variable domain (VL2); and a second heavy chain variable domain (VH2); wherein the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively. In some embodiments, the bispecific antibodies provided herein have two identical LC and HC pairs.

[0139] Alternatively, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19, comprising (1) a light chain (LC) comprising a first light chain variable domain (VL1), a CL region, a second light chain variable domain (VL2), and a second heavy chain variable domain (VH2); and (2) a heavy chain (HC) comprising a first heavy chain variable domain (VH1) and a heavy chain constant region (CH); wherein the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively. In some embodiments, the bispecific antibodies provided herein have two identical LC and HC pairs.

[0140] Alternatively, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19, comprising (1) a light chain (LC) comprising a first light chain variable domain (VL1) and a CL region; and (2) a heavy chain (HC) comprising a first heavy chain variable domain (VH1), a heavy chain constant region (CH), a second heavy chain variable domain (VH2); and a second light chain variable domain (VL2); wherein the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively. In some embodiments, the bispecific antibodies provided herein have two identical LC and HC pairs.

[0141] Alternatively, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19, comprising (1) a light chain (LC) comprising a first light chain variable domain (VL1), a CL region, a second heavy chain variable domain (VH2), and a second light chain variable domain (VL2); and (2) a heavy chain (HC) comprising a first heavy chain variable domain (VH1) and a heavy chain constant region (CH); wherein the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively. In some embodiments, the bispecific antibodies provided herein have two identical LC and HC pairs.

[0142] The amino acid sequences of the CL and CH regions of the bispecific antibodies disclosed herein can be derived from any suitable source, e.g., the constant regions of antibodies such as the constant regions of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG1. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG2. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG3. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG4. In some embodiments, the amino acid sequences of the CH1, CL regions, and Fc regions (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein can contain one or more amino acid substitutions that are different from the wild-type immunoglobulin, e.g., one or more amino acid substitutions in wild-type IgG1 or IgG4. These substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US 8323962, US6737056, and US7416727).

[0143] In some embodiments, the CH region can be selected from the human IgG1 CH region (SEQ ID NO:58), the human IgG2 CH region (SEQ ID NO:80), the human IgG3 CH region (SEQ ID NO:81), and the human IgG4 CH region (SEQ ID NO:82). In some embodiments, the CH region is the human IgG1 CH region (SEQ ID NO:58).

[0144] In some embodiments, the CL region can be κCL (Cκ; SEQ ID NO:68). In some embodiments, the CL region can be λCL (Cλ, SEQ ID NO:69).

[0145] In some embodiments of the bispecific antibodies provided herein, (1) the CL region is Cκ (SEQ ID NO:68) or Cλ (SEQ ID NO:69); or (2) the CH region is the human IgG1 CH region (SEQ ID NO:58); or both (1) and (2).

[0146] In some embodiments of the bispecific antibodies provided herein, the scFv is linked to the CH or CL region via a linker. In some embodiments, the linker has the amino acid sequence (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO:71). In some embodiments, the linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:72). In some embodiments, the linker has the amino acid sequence (EAAAK)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO:73).

[0147] In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 of the scFv are linked via a linker. In some embodiments, the linker has the amino acid sequence (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO:71). In some embodiments, the linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:72). In some embodiments, the linker has the amino acid sequence (EAAAK)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO:73).

[0148] In some embodiments of the bispecific antibodies provided herein, the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19. The VL1 / VH1 pair can be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL1 and VH1 are the VL and VH of adalimumab and have the amino acid sequences shown by SEQ ID NO:1 and 2, respectively. In some embodiments, VL1 and VH1 are the VL and VH of golimumab and have the amino acid sequences shown by SEQ ID NO:5 and 6, respectively. The VL2 / VH2 pair can be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL2 and VH2 are the VL and VH of guselkumab and have the amino acid sequences shown by SEQ ID NO:10 and 11, respectively. In some embodiments, VL2 and VH2 are the VL and VH of a guselkumab variant and have the amino acid sequences shown by SEQ ID NO:93 and 94, respectively. In some embodiments, VL2 and VH2 are the VL and VH of tildrakizumab and have the amino acid sequences shown by SEQ ID NO:14 and 15, respectively.

[0149] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have (1) amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) amino acid sequences shown by SEQ ID NO: 5 and 6 respectively. In some embodiments, VL1 and VH1 have amino acid sequences shown by SEQ ID NO: 1 and 2 respectively. In some embodiments, VL1 and VH1 have amino acid sequences shown by SEQ ID NO: 5 and 6 respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) amino acid sequences shown by SEQ ID NO: 14 and 15 respectively; or (3) amino acid sequences shown by SEQ ID NO: 93 and 94 respectively. In some embodiments, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 10 and 11 respectively. In some embodiments, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 14 and 15 respectively. In some embodiments, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 93 and 94 respectively.

[0150] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 1, 2, 10 and 11 respectively; (2) amino acid sequences shown by SEQ ID NO: 1, 2, 14 and 15 respectively; (3) amino acid sequences shown by SEQ ID NO: 1, 2, 93 and 94 respectively; (4) amino acid sequences shown by SEQ ID NO: 5, 6, 10 and 11 respectively; (5) amino acid sequences shown by SEQ ID NO: 5, 6, 14 and 15 respectively; or (6) amino acid sequences shown by SEQ ID NO: 5, 6, 93 and 94 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 1, 2, 10 and 11 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 1, 2, 14 and 15 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 1, 2, 93 and 94 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 5, 6, 10 and 11 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 5, 6, 14 and 15 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 5, 6, 93 and 94 respectively.

[0151] In some embodiments of the bispecific antibodies provided herein, the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα. The VL1 / VH1 pair can be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL1 and VH1 are the VL and VH of guselkumab and have the amino acid sequences shown by SEQ ID NO:10 and 11, respectively. In some embodiments, VL1 and VH1 are the VL and VH of tildrakizumab and have the amino acid sequences shown by SEQ ID NO:14 and 15, respectively. The VL2 / VH2 pair can be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL2 and VH2 are the VL and VH of adalimumab and have the amino acid sequences shown by SEQ ID NO:1 and 2, respectively. In some embodiments, VL2 and VH2 are the VL and VH of golimumab and have the amino acid sequences shown by SEQ ID NO:5 and 6, respectively.

[0152] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO:10 and 11, respectively; or (2) the amino acid sequences shown by SEQ ID NO:14 and 15, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown by SEQ ID NO:10 and 11, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown by SEQ ID NO:14 and 15, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO:1 and 2, respectively; or (2) the amino acid sequences shown by SEQ ID NO:5 and 6, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown by SEQ ID NO:1 and 2, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown by SEQ ID NO:5 and 6, respectively.

[0153] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have (1) amino acid sequences shown by SEQ ID NO: 10, 11, 1, and 2, respectively; (2) amino acid sequences shown by SEQ ID NO: 10, 11, 5, and 6, respectively; (3) amino acid sequences shown by SEQ ID NO: 14, 15, 1, and 2, respectively; or (4) amino acid sequences shown by SEQ ID NO: 14, 15, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 10, 11, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 10, 11, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 14, 15, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 14, 15, 5, and 6, respectively. The following provides a list of exemplary LCs and HCs of bispecific antibodies as Table 4B.

[0154] Table 4B: Exemplary bispecific antibodies against human TNFα and human IL23p19

[0155] In some embodiments, provided herein are bispecific antibodies with an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with (1) amino acid sequences shown by SEQ ID NO: 3 and 30, respectively; (2) amino acid sequences shown by SEQ ID NO: 3 and 31, respectively; (3) amino acid sequences shown by SEQ ID NO: 7 and 32, respectively; (4) amino acid sequences shown by SEQ ID NO: 7 and 33, respectively; (5) amino acid sequences shown by SEQ ID NO: 12 and 34, respectively; (6) amino acid sequences shown by SEQ ID NO: 12 and 35, respectively; (7) amino acid sequences shown by SEQ ID NO: 16 and 36, respectively; (8) amino acid sequences shown by SEQ ID NO: 16 and 37, respectively; or (9) amino acid sequences shown by SEQ ID NO: 7 and 92, respectively.

[0156] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequences shown by SEQ ID NO:3 and 30, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:3. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO:3. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:30. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO:30. In some embodiments, the LC and the HC have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO:3 and 30, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO:3 and 30, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO:3 and 30, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO:3 and 30, respectively. In some embodiments, provided herein is a bispecific antibody designated as B1, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO:3 and 30, respectively.

[0157] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 3 and 31, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 3. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 3. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 31. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 31. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 3 and 31, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 3 and 31, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 3 and 31, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 3 and 31, respectively. In some embodiments, provided herein is a bispecific antibody designated as B2, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 3 and 31, respectively.

[0158] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 32, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:7. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO:7. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:32. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO:32. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 32, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 32, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 32, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 32, respectively. In some embodiments, provided herein is a bispecific antibody designated as B3, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO:7 and 32, respectively.

[0159] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequences shown by SEQ ID NO:7 and 92, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:7. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO:7. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:92. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO:92. In some embodiments, the LC and the HC have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO:7 and 92, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO:7 and 92, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO:7 and 92, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO:7 and 92, respectively. In some embodiments, provided herein is a bispecific antibody designated as B3-1, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO:7 and 92, respectively.

[0160] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 33, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:7. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO:7. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:33. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO:33. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 33, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 33, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 33, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO:7 and 33, respectively. In some embodiments, provided herein is a bispecific antibody designated as B4, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO:7 and 33, respectively.

[0161] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 34, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 12. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 12. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 34. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 34. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 34, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 34, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 34, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 34, respectively. In some embodiments, provided herein is a bispecific antibody designated as B5, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 12 and 34, respectively.

[0162] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 35, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 12. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 12. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 35. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 35. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 35, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 35, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 35, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 35, respectively. In some embodiments, provided herein is a bispecific antibody designated as B6, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 12 and 35, respectively.

[0163] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 36, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 16. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 16. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 36. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 36. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 36, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 36, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 36, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 36, respectively. In some embodiments, provided herein is a bispecific antibody designated as B7, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 16 and 36, respectively.

[0164] In some embodiments, provided herein are bispecific antibodies with an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequences shown by SEQ ID NO: 16 and 37, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 16. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 37. In some embodiments, the LC and the HC have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO: 16 and 37, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO: 16 and 37, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO: 16 and 37, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO: 16 and 37, respectively. In some embodiments, provided herein is a bispecific antibody designated B8, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 16 and 37, respectively. 2.4 DVD-Ig

[0165] In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and bind to human IL23p19 have Figure 1C the "dual variable domain - immunoglobulin" or "DVD-Ig" structure shown in. The DVD-Ig is a symmetric structure with four antigen-binding sites that can simultaneously target two different targets. The DVD-Ig structure contains an Fc region, and each antibody arm uses a flexible short peptide to link the two variable regions.

[0166] Accordingly, provided herein are bispecific antibodies that specifically bind to human TNFα and bind to human IL23p19, comprising (1) a light chain (LC) comprising a first light chain variable domain (VL1), a second light chain variable domain (VL2), and a CL region; and (2) a heavy chain (HC) comprising a first heavy chain variable domain (VH1), a second heavy chain variable domain (VH2), and a CH region; wherein the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively.

[0167] The amino acid sequences of the CL and CH regions of the bispecific antibodies disclosed herein can be derived from any suitable source, e.g., the constant regions of antibodies such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG1. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG2. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG3. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG4. In some embodiments, the amino acid sequences of the CH1, CL regions, and Fc regions (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein can comprise one or more amino acid substitutions that are different from the wild-type immunoglobulin, e.g., one or more amino acid substitutions in wild-type IgG1 or IgG4. Such substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US 8323962, US6737056, and US7416727).

[0168] In some embodiments, the CH region can be selected from the human IgG1 CH region (SEQ ID NO:58), the human IgG2 CH region (SEQ ID NO:80), the human IgG3 CH region (SEQ ID NO:81), and the human IgG4 CH region (SEQ ID NO:82). In some embodiments, the CH region is the human IgG1 CH region (SEQ ID NO:58).

[0169] In some embodiments, the CL region can be κCL (Cκ; SEQ ID NO:68). In some embodiments, the CL region can be λCL (Cλ, SEQ ID NO:69).

[0170] In some embodiments of the bispecific antibodies provided herein, (1) the CL region is Cκ (SEQ ID NO: 68) or Cλ (SEQ ID NO: 69); or (2) the CH region is a human IgG1 CH region (SEQ ID NO: 58); or both (1) and (2).

[0171] In some embodiments of the bispecific antibodies provided herein, the variable domains are linked together via a linker. In some embodiments, VH1 is linked to VH2 via a linker. In some embodiments, VL1 is linked to VL2 via a linker. In some embodiments, the linker has the amino acid sequence (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 71). In some embodiments, the linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 72). In some embodiments, the linker has the amino acid sequence (EAAAK)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 73).

[0172] In some embodiments of the bispecific antibodies provided herein, the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19. The VL1 / VH1 pair can be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL1 and VH1 are the VL and VH of adalimumab and have the amino acid sequences shown by SEQ ID NO: 1 and 2, respectively. In some embodiments, VL1 and VH1 are the VL and VH of golimumab and have the amino acid sequences shown by SEQ ID NO: 5 and 6, respectively. The VL2 / VH2 pair can be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL2 and VH2 are the VL and VH of guselkumab and have the amino acid sequences shown by SEQ ID NO: 10 and 11, respectively. In some embodiments, VL2 and VH2 are the VL and VH of tildrakizumab and have the amino acid sequences shown by SEQ ID NO: 14 and 15, respectively.

[0173] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have (1) amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) amino acid sequences shown by SEQ ID NO: 5 and 6 respectively. In some embodiments, VL1 and VH1 have amino acid sequences shown by SEQ ID NO: 1 and 2 respectively. In some embodiments, VL1 and VH1 have amino acid sequences shown by SEQ ID NO: 5 and 6 respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) amino acid sequences shown by SEQ ID NO: 14 and 15 respectively. In some embodiments, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 10 and 11 respectively. In some embodiments, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 14 and 15 respectively.

[0174] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 1, 2, 10 and 11 respectively; (2) amino acid sequences shown by SEQ ID NO: 1, 2, 14 and 15 respectively; (3) amino acid sequences shown by SEQ ID NO: 5, 6, 10 and 11 respectively; or (4) amino acid sequences shown by SEQ ID NO: 5, 6, 14 and 15 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 1, 2, 10 and 11 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 1, 2, 14 and 15 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 5, 6, 10 and 11 respectively. In some embodiments, VL1, VH1, VL2 and VH2 have amino acid sequences shown by SEQ ID NO: 5, 6, 14 and 15 respectively.

[0175] In some embodiments of the bispecific antibodies provided herein, the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα. The VL1 / VH1 pair can be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL1 and VH1 are the VL and VH of guselkumab and have the amino acid sequences shown by SEQ ID NO:10 and 11, respectively. In some embodiments, VL1 and VH1 are the VL and VH of tildrakizumab and have the amino acid sequences shown by SEQ ID NO:14 and 15, respectively. The VL2 / VH2 pair can be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL2 and VH2 are the VL and VH of adalimumab and have the amino acid sequences shown by SEQ ID NO:1 and 2, respectively. In some embodiments, VL2 and VH2 are the VL and VH of golimumab and have the amino acid sequences shown by SEQ ID NO:5 and 6, respectively.

[0176] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO:10 and 11, respectively; or (2) the amino acid sequences shown by SEQ ID NO:14 and 15, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown by SEQ ID NO:10 and 11, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown by SEQ ID NO:14 and 15, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO:1 and 2, respectively; or (2) the amino acid sequences shown by SEQ ID NO:5 and 6, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown by SEQ ID NO:1 and 2, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown by SEQ ID NO:5 and 6, respectively.

[0177] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have (1) amino acid sequences shown by SEQ ID NO: 10, 11, 1, and 2, respectively; (2) amino acid sequences shown by SEQ ID NO: 10, 11, 5, and 6, respectively; (3) amino acid sequences shown by SEQ ID NO: 14, 15, 1, and 2, respectively; or (4) amino acid sequences shown by SEQ ID NO: 14, 15, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 10, 11, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 10, 11, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 14, 15, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have amino acid sequences shown by SEQ ID NO: 14, 15, 5, and 6, respectively. The following provides a list of exemplary LCs and HCs of bispecific antibodies as Table 4C.

[0178] Table 4C: Exemplary bispecific antibodies against human TNFα and human IL23p19

[0179] In some embodiments, provided herein are bispecific antibodies with LC and HC that specifically bind to human TNFα and bind to human IL23p19, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with (1) amino acid sequences shown by SEQ ID NO: 38 and 39, respectively; (2) amino acid sequences shown by SEQ ID NO: 40 and 41, respectively; (3) amino acid sequences shown by SEQ ID NO: 42 and 43, respectively; (4) amino acid sequences shown by SEQ ID NO: 44 and 45, respectively; (5) amino acid sequences shown by SEQ ID NO: 46 and 47, respectively; (6) amino acid sequences shown by SEQ ID NO: 48 and 49, respectively; (7) amino acid sequences shown by SEQ ID NO: 50 and 51, respectively; or (8) amino acid sequences shown by SEQ ID NO: 52 and 53, respectively.

[0180] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequences shown by SEQ ID NO: 38 and 39, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 38. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 39. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 39. In some embodiments, the LC and the HC have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO: 38 and 39, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO: 38 and 39, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO: 38 and 39, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO: 38 and 39, respectively. In some embodiments, provided herein is a bispecific antibody designated as C1, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 38 and 39, respectively.

[0181] In some embodiments, provided herein are bispecific antibodies with an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 40. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 40. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 41. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 41. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, provided herein is a bispecific antibody designated as C2, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 40 and SEQ ID NO: 41, respectively.

[0182] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 42. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 42. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 43. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 43. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, provided herein is a bispecific antibody designated as C3, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0183] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequences shown by SEQ ID NO: 44 and SEQ ID NO: 45, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 44. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 45. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 45. In some embodiments, the LC and the HC have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO: 44 and SEQ ID NO: 45, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO: 44 and SEQ ID NO: 45, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO: 44 and SEQ ID NO: 45, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO: 44 and SEQ ID NO: 45, respectively. In some embodiments, provided herein is a bispecific antibody designated as C4, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 44 and SEQ ID NO: 45, respectively.

[0184] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 46 and 47, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 46. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 46. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 47. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 47. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 46 and 47, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 46 and 47, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 46 and 47, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 46 and 47, respectively. In some embodiments, provided herein is a bispecific antibody designated as C5, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 46 and 47, respectively.

[0185] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 48 and SEQ ID NO: 49, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 48. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 48. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 49. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 49. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 48 and SEQ ID NO: 49, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 48 and SEQ ID NO: 49, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 48 and SEQ ID NO: 49, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 48 and SEQ ID NO: 49, respectively. In some embodiments, provided herein is a bispecific antibody designated as C6, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 48 and SEQ ID NO: 49, respectively.

[0186] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 50 and 51, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 50. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 50. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 51. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 51. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 50 and 51, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 50 and 51, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 50 and 51, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 50 and 51, respectively. In some embodiments, provided herein is a bispecific antibody designated as C7, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 50 and 51, respectively.

[0187] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequences shown by SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:52. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO:52. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:53. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO:53. In some embodiments, the LC and the HC have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, provided herein is a bispecific antibody designated as C8, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO:52 and SEQ ID NO:53, respectively. 2.5SMAB

[0188] In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and bind to human IL23p19 have Figure 1D the "single domain antibody fused to a monoclonal antibody" or "SMAB" structure shown. The single domain antibody can be a single heavy chain variable domain antibody ("VHH"). The VHH can be linked to the heavy chain or the light chain of an IgG. In some embodiments, the VHH is linked to the heavy chain of an IgG. In some embodiments, the VHH is linked to the N-terminus of the heavy chain of an IgG. In some embodiments, the VHH is linked to the C-terminus of the heavy chain of an IgG. In some embodiments, the VHH is linked to the light chain of an IgG. In some embodiments, the VHH is linked to the N-terminus of the light chain of an IgG. In some embodiments, the VHH is linked to the C-terminus of the light chain of an IgG.

[0189] Accordingly, the present invention provides bispecific antibodies that specifically bind to human TNFα and human IL23p19, comprising: (1) a light chain (LC) comprising a light chain variable domain (VL) and a CL region; and (2) a heavy chain (HC) comprising a heavy chain variable domain (VH) and a CH region; wherein (1) the VL / VH pair specifically binds to human IL23p19; and (2) the HC further comprises a VHH that specifically binds to human TNFα. In some embodiments, the VHH is linked to the N-terminus of the HC. In some embodiments, the VHH is linked to the C-terminus of the HC.

[0190] The present invention also provides bispecific antibodies that specifically bind to human TNFα and human IL23p19, comprising: (1) a light chain (LC) comprising a light chain variable domain (VL) and a CL region; and (2) a heavy chain (HC) comprising a heavy chain variable domain (VH) and a CH region; wherein (1) the VL / VH pair specifically binds to human TNFα; and (2) the HC further comprises a VHH that specifically binds to human IL23p19. In some embodiments, the VHH is linked to the N-terminus of the HC. In some embodiments, the VHH is linked to the C-terminus of the HC.

[0191] The present invention also provides bispecific antibodies that specifically bind to human TNFα and human IL23p19, comprising: (1) a light chain (LC) comprising a light chain variable domain (VL) and a CL region; and (2) a heavy chain (HC) comprising a heavy chain variable domain (VH) and a CH region; wherein (1) the VL / VH pair specifically binds to human IL23p19; and (2) the LC further comprises a VHH that specifically binds to human TNFα. In some embodiments, the VHH is linked to the N-terminus of the HC. In some embodiments, the VHH is linked to the C-terminus of the LC.

[0192] The present invention also provides bispecific antibodies that specifically bind to human TNFα and human IL23p19, comprising: (1) a light chain (LC) comprising a light chain variable domain (VL) and a CL region; and (2) a heavy chain (HC) comprising a heavy chain variable domain (VH) and a CH region; wherein (1) the VL / VH pair specifically binds to human TNFα; and (2) the LC further comprises a VHH that specifically binds to human IL23p19. In some embodiments, the VHH is linked to the N-terminus of the LC. In some embodiments, the VHH is linked to the C-terminus of the LC.

[0193] The amino acid sequences of the CL and CH regions of the bispecific antibodies disclosed herein can be derived from any suitable source, e.g., the constant regions of antibodies such as IgG1, IgG2, IgG3 or IgG4. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG1. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG2. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG3. In some embodiments, the constant domains and constant regions of the bispecific antibodies provided herein are derived from human IgG4. In some embodiments, the amino acid sequences of the CH1, CL regions and Fc regions (hinge, CH2 and CH3) of the bispecific antibodies disclosed herein may comprise one or more amino acid substitutions different from wild-type immunoglobulins, e.g., one or more amino acid substitutions in wild-type IgG1 or IgG4. Such substitutions are known in the art (see, e.g., US7704497, US7083784, US6821505, US 8323962, US6737056 and US7416727).

[0194] In some embodiments, the CH region can be selected from the human IgG1 CH region (SEQ ID NO:58), the human IgG2 CH region (SEQ ID NO:80), the human IgG3 CH region (SEQ ID NO:81) and the human IgG4 CH region (SEQ ID NO:82). In some embodiments, the CH region is the human IgG1 CH region (SEQ ID NO:58).

[0195] In some embodiments, the CL region can be κCL (Cκ; SEQ ID NO:68). In some embodiments, the CL region can be λCL (Cλ, SEQ ID NO:69).

[0196] In some embodiments of the bispecific antibodies provided herein, (1) the CL region is Cκ (SEQ ID NO:68) or Cλ (SEQ ID NO:69); or (2) the CH region is the human IgG1 CH region (SEQ ID NO:58); or both (1) and (2).

[0197] In some embodiments of the bispecific antibodies provided herein, the VHH is linked to the IgG heavy chain via a linker. In some embodiments, the C-terminus of the VHH is linked to the N-terminus of the VH via a linker. In some embodiments, the N-terminal VHH is linked to the C-terminus of the CH region via a linker. In some embodiments of the bispecific antibodies provided herein, the VHH is linked to the IgG light chain via a linker. In some embodiments, the C-terminus of the VHH is linked to the N-terminus of the VL via a linker. In some embodiments, the N-terminal VHH is linked to the C-terminus of the CL region via a linker. In some embodiments, the linker has the amino acid sequence (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO:71). In some embodiments, the linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:72). In some embodiments, the linker has the amino acid sequence (EAAAK)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO:73).

[0198] In some embodiments, the bispecific antibodies provided herein comprise (1) a light chain (LC) comprising a light chain variable domain (VL) and a CL region; and (2) a heavy chain (HC) comprising a heavy chain variable domain (VH) and a CH region; wherein (1) the VL / VH pair specifically binds to human IL23p19; and (2) the HC further comprises a VHH that specifically binds to human TNFα. In some embodiments, the VHH is linked to the N-terminus of the HC. In some embodiments, the VHH is linked to the C-terminus of the HC. The VL / VH pair can be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, the VL and VH are the VL and VH of guselkumab and have the amino acid sequences shown by SEQ ID NO:10 and 11, respectively. In some embodiments, the VL and VH are the VL and VH of tildrakizumab and have the amino acid sequences shown by SEQ ID NO:14 and 15, respectively. The VHH can be any VHH that specifically binds to human TNFα. In some embodiments, the VHH can be ozoralizumab (SEQ ID NO:9).

[0199] In some embodiments of the bispecific antibodies provided herein, VL and VH have amino acid sequences shown by SEQ ID NO: 10 and 11 respectively, and VHH has the amino acid sequence shown by SEQ ID NO: 9. In some embodiments, VL and VH have amino acid sequences shown by SEQ ID NO: 14 and 15 respectively, and VHH has the amino acid sequence shown by SEQ ID NO: 9. The following is a list of exemplary LCs and HCs of the bispecific antibodies provided as Table 4D.

[0200] Table 4D: Exemplary Bispecific Antibodies Against Human TNFα and Human IL23p19

[0201] In some embodiments, provided herein are bispecific antibodies with an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 54 respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 12. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 12. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 54. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 54. In some embodiments, the LC and HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 54 respectively. In some embodiments, the LC and HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 54 respectively. In some embodiments, the LC and HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 54 respectively. In some embodiments, the LC and HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 12 and 54 respectively. In some embodiments, provided herein is a bispecific antibody designated as D1, wherein the LC and HC have the amino acid sequences shown by SEQ ID NO: 12 and 54 respectively.

[0202] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequences shown by SEQ ID NO: 12 and 55, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 12. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 55. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 55. In some embodiments, the LC and the HC have at least 90% sequence identity to the amino acid sequences shown by SEQ ID NO: 12 and 55, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity to the amino acid sequences shown by SEQ ID NO: 12 and 55, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity to the amino acid sequences shown by SEQ ID NO: 12 and 55, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity to the amino acid sequences shown by SEQ ID NO: 12 and 55, respectively. In some embodiments, provided herein is a bispecific antibody designated as D2, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 12 and 55, respectively.

[0203] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 56, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 16. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 16. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 56. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 56. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 56, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 56, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 56, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 56, respectively. In some embodiments, provided herein is a bispecific antibody designated as D3, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 16 and 56, respectively.

[0204] In some embodiments, provided herein are bispecific antibodies having an LC and an HC that specifically bind to human TNFα and bind to human IL23p19, wherein the LC and the HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 57, respectively. In some embodiments, the LC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 16. In some embodiments, the LC has the amino acid sequence shown by SEQ ID NO: 16. In some embodiments, the HC has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 57. In some embodiments, the HC has the amino acid sequence shown by SEQ ID NO: 57. In some embodiments, the LC and the HC have at least 90% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 57, respectively. In some embodiments, the LC and the HC have at least 95% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 57, respectively. In some embodiments, the LC and the HC have at least 98% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 57, respectively. In some embodiments, the LC and the HC have at least 99% sequence identity with the amino acid sequences shown by SEQ ID NO: 16 and 57, respectively. In some embodiments, provided herein is a bispecific antibody designated as D4, wherein the LC and the HC have the amino acid sequences shown by SEQ ID NO: 16 and 57, respectively. 2.6 Variants

[0205] The present disclosure also contemplates other variants and equivalent forms that are substantially homologous to the bispecific antibodies described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including (but not limited to) specificity, thermal stability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites or altering the membrane anchoring characteristics.

[0206] The present invention also provides antibodies comprising functional variants of the heavy chain, light chain, VL region, VH region, or one or more CDRs of the antibodies described in the examples. Functional variants of the heavy chain, light chain, VL, VH, or CDR used in the context of an antibody still allow the antibody to retain at least a significant proportion (at least about 90%, 95% or more) of the functional characteristics of the "reference" and / or "parental" antibody, including affinity and / or specificity / selectivity, Fc inertness, and PK parameters such as half-life, Tmax, Cmax. These functional variants generally retain significant sequence identity to the parental antibody and / or have substantially similar lengths of the heavy and light chains. Exemplary variants include those of the heavy chain and / or light chain, VH and / or VL, and / or CDR regions that differ from the parental antibody sequence primarily by conservative substitutions. For example, 10, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution(s) in the variant can be conservative amino acid residue substitutions.

[0207] In some embodiments, variants of the bispecific antibodies disclosed herein can retain their ability to bind TNFα and IL23p19 to a similar extent, the same extent, or a higher extent as the parental bispecific antibody. In some embodiments, the variant can be at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher identical to the parental antibody or antigen-binding fragment in terms of amino acid sequence. In certain embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parental bispecific antibodies disclosed herein having one or more conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having a particular physical and / or chemical property is replaced with another amino acid having the same or similar chemical or physical property.

[0208] In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parental antibody having one or more non-conservative amino acid substitutions. In some embodiments, variants of the bispecific antibodies disclosed herein comprise the amino acid sequence of the parental binding antibody having one or more non-conservative amino acid substitutions, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant. In certain embodiments, the one or more conservative amino acid substitutions and / or the one or more non-conservative amino acid substitutions can enhance the biological activity of the variant such that the biological activity of the functional variant is increased compared to the parental antibody.

[0209] In some embodiments, the variant can have 1, 2, 3, 4, or 5 amino acid substitutions in the CDRs of the binding portion (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3).

[0210] In some embodiments, the bispecific antibodies provided herein include modifications in their Fc regions. In some embodiments, the modified antibodies (e.g., modified Fc regions) provide altered effector functions, which in turn affect the biological profile of the antibody. For example, in some embodiments, deletion or inactivation (by point mutation or otherwise) of the constant region reduces Fc receptor binding of the modified antibody when it circulates. In some embodiments, the constant region modification reduces the immunogenicity of the antibody. In some embodiments, the constant region modification increases the serum half-life of the antibody. In some embodiments, the constant region modification reduces the serum half-life of the antibody. In some embodiments, the constant region modification reduces or removes the antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acid substitutions in the human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduce the effector functions (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody does not have one or more effector functions (e.g., an “effectorless” antibody). In some embodiments, the antibody has no ADCC activity and / or no CDC activity. In some embodiments, the antibody does not bind Fc receptors and / or complement factors. In some embodiments, the antibody does not have effector functions. In some embodiments, the constant region modification increases or enhances the ADCC and / or CDC of the antibody. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified to add / replace one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.

[0211] In some embodiments of the bispecific antibodies provided herein, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors. The Fc receptor can be a human Fc receptor. The Fc receptor can be an Fcγ receptor. The Fc receptor can be an activating Fc receptor. The Fc receptor can be an activating human Fcγ receptor, such as human FcγRIIIa, FcγRI or FcγRIIa. In some embodiments of the bispecific antibodies provided herein, the Fc domain comprises one or more amino acid substitutions that reduce effector function. The effector function can be complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, or any combination thereof. In some embodiments, the effector function is ADCC.

[0212] In some embodiments of the bispecific antibodies provided herein, the same one or more amino acid substitutions are present in each of the two subunits of the Fc region. In one aspect, the one or more amino acid substitutions reduce the binding affinity of the Fc region for Fc receptors. In one aspect, the one or more amino acid substitutions reduce the binding affinity of the Fc region for Fc receptors by at least 2-fold, at least 5-fold, or at least 10-fold.

[0213] In some embodiments, the Fc region of the bispecific antibodies provided herein comprises amino acid substitutions at positions selected from E233, L234, L235, N297, P331, and P329. In some embodiments, the Fc region comprises amino acid substitutions at positions selected from L234, L235, and P329. In some embodiments, the Fc region comprises the amino acid substitutions L234A and L235A. In some embodiments, the Fc region is an IgG1 Fc region, specifically a human IgG1 Fc region. In some embodiments, the Fc region comprises an amino acid substitution at position P329, and in some embodiments, the amino acid substitution is P329A or P329G. In some embodiments, the Fc region comprises an amino acid substitution at position P329 and further comprises amino acid substitutions at positions selected from E233, L234, L235, N297, and P331. In some embodiments, the other amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In some embodiments, the Fc region comprises amino acid substitutions at positions P329, L234, and L235. In a more specific aspect, the Fc region comprises the amino acid mutations L234A, L235A, and P329G. In some embodiments, the Fc region comprises the amino acid substitutions L234A, L235A, and P329G. All amino acid residues are numbered according to the EU index.

[0214] In some embodiments, variants can include additions of amino acid residues at the amino and / or carboxyl termini of the antibody. The length of other amino acid residues can range from 1 residue to 100 or more residues. In some embodiments, the variant contains an N-terminal methionyl residue. In some embodiments, the variant is engineered to be detectable and can contain a detectable label and / or protein (e.g., a fluorescent tag or an enzyme).

[0215] The variant antibodies described herein can be produced using methods known in the art, including (but not limited to) site-directed mutagenesis, alanine-scanning mutagenesis, and PCR mutagenesis.

[0216] In some embodiments, the bispecific antibodies disclosed herein can be modified naturally or by intervening chemical modification. In some embodiments, the bispecific antibody is chemically modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, and / or conjugation to a cell ligand or other protein. Any of a variety of chemical modifications can be carried out by known techniques. The bispecific antibodies provided herein can contain one or more analogs of amino acids (including, for example, unnatural amino acids) and other modifications known in the art.

[0217] The bispecific antibodies disclosed in the present invention can be analyzed for their physical, chemical, and / or biological properties by a variety of methods known in the art. In some embodiments, the bispecific antibodies provided herein are tested for their ability to bind human TNFα and / or human IL23p19. Binding assays include (but are not limited to) BLI, SPR (e.g., Biacore), ELISA, and FACS. Additionally, the solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency of the antibody can be evaluated.

[0218] In some embodiments, the bispecific antibodies disclosed herein can be conjugated to a detectable substance or molecule, which allows the reagent to be used for detection. Detectable substances can include (but are not limited to) enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; cofactors such as biotin and flavin; fluorescent substances such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials such as luciferase; radioactive substances such as 212 Bi, 14 C, 57 Co, 51 Cr, 67 Cu, 18 F, 68 Ga,67 Ga, 153 Gd, 159 Gd, 68 Ge, 3 H, 166 Ho, 131 I, 125 I, 123 I, 121 I, 115 In, 113 In, 112 In, 111 In, 140 La, 177 Lu, 54 Mn, 99 Mo, 32 P, 103 Pd, 149 Pm, 142 Pr, 186 Re, 188 Re, 105 Rh, 97 Ru, 35 S, 47 Sc, 75 Se, 153 Sm, 113 Sn, 117 Sn, 85 Sr, 99m Tc, 201 Ti, 133 Xe, 90 Y, 69 Yb, 175 Yb, 65 Zn; a positron-emitting metal; and a magnetic metal ion a positron-emitting metal; and a magnetic metal ion.

[0219] The anti-TNFa / IL23p19 bispecific antibodies disclosed herein can be linked to a solid support. These solid supports include (but are not limited to) glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized bispecific antibody is used in an immunoassay. In some embodiments, the immobilized bispecific antibody is used in purification. 3. Polynucleotides, vectors and cells

[0220] The present disclosure provides polynucleotides encoding at least one light chain or one heavy chain of an anti-TNFα / IL23p19 bispecific antibody disclosed herein. In some embodiments, the polynucleotides provided herein encode a polypeptide of the bispecific antibody, such as a light chain or a heavy chain. In some embodiments, the polynucleotides provided herein encode more than one polypeptide. In some embodiments, the polynucleotides provided herein can encode, for example, the light chain and the heavy chain of the bispecific antibody provided herein. The cistrons can be separated by, for example, an internal ribosome entry site (IRES) or a 2A element. As understood in the art, an IRES refers to a nucleotide sequence in an expression cassette that, when transcribed into mRNA, can directly recruit ribosomes without a prior scan of the untranslated region of the mRNA by the ribosomes. As understood in the art, a 2A element encoding a self-cleaving short peptide (about 20 amino acids) provides a mechanism for the subsequent separation of equimolar amounts of the polypeptides of interest. Exemplary 2A self-cleaving peptides include P2A (SEQ ID NO:76), E2A (SEQ ID NO:77), F2A (SEQ ID NO:78), and T2A (SEQ ID NO:79).

[0221] In some embodiments, the present disclosure provides polynucleotides encoding LC1, HC1, LC2, HC2, or any combination thereof of an anti-TNFα / IL23p19 bispecific antibody disclosed herein having a CrossMab-KIH structure. In some embodiments, the present disclosure provides polynucleotides encoding LC, HC, or both of an anti-TNFα / IL23p19 bispecific antibody disclosed herein having an IgG-ScFv structure, a DVD-Ig structure, or a SMAB structure.

[0222] As used herein, the term “encoding” and its grammatical equivalents refer to the inherent property of a specific nucleotide sequence in a polynucleotide or nucleic acid, such as a gene, cDNA, or mRNA, to serve as a template in a biological process for synthesizing a polymer and macromolecule having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate to one another and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs can include introns.

[0223] In some embodiments, provided herein are polynucleotides encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A1, or any combination thereof. In some embodiments, provided herein are multiple polynucleotides co-encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A1. In some embodiments, provided herein are polynucleotides encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A2, or any combination thereof. In some embodiments, provided herein are multiple polynucleotides co-encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A2. In some embodiments, provided herein are polynucleotides encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A3, or any combination thereof. In some embodiments, provided herein are multiple polynucleotides co-encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A3. In some embodiments, provided herein are polynucleotides encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A4, or any combination thereof. In some embodiments, provided herein are multiple polynucleotides co-encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A4. In some embodiments, provided herein are polynucleotides encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A5, or any combination thereof. In some embodiments, provided herein are multiple polynucleotides co-encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A5. In some embodiments, provided herein are polynucleotides encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A6, or any combination thereof. In some embodiments, provided herein are multiple polynucleotides co-encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A6. In some embodiments, provided herein are polynucleotides encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A7, or any combination thereof. In some embodiments, provided herein are multiple polynucleotides co-encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A7. In some embodiments, provided herein are polynucleotides encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A8, or any combination thereof. In some embodiments, provided herein are multiple polynucleotides co-encoding LC1, HC1, LC2, and HC2 of a bispecific antibody designated as A8.

[0224] In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as B1. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as B1. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as B2. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as B2. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as B3. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as B3. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as B4. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as B4. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as B5. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as B5. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as B6. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as B6. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as B7. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as B7. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as B8. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as B8.

[0225] In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as C1. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as C1. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as C2. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as C2. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as C3. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as C3. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as C4. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as C4. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as C5. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as C5. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as C6. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as C6. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as C7. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as C7. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both of a bispecific antibody designated as C8. In some embodiments, provided herein are a first and a second polynucleotide encoding the LC and HC, respectively, of a bispecific antibody designated as C8.

[0226] In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both, of a bispecific antibody designated as D1. In some embodiments, provided herein are first and second polynucleotides encoding the LC and HC, respectively, of a bispecific antibody designated as D1. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both, of a bispecific antibody designated as D2. In some embodiments, provided herein are first and second polynucleotides encoding the LC and HC, respectively, of a bispecific antibody designated as D2. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both, of a bispecific antibody designated as D3. In some embodiments, provided herein are first and second polynucleotides encoding the LC and HC, respectively, of a bispecific antibody designated as D3. In some embodiments, provided herein are polynucleotides encoding the LC, HC, or both, of a bispecific antibody designated as D4. In some embodiments, provided herein are first and second polynucleotides encoding the LC and HC, respectively, of a bispecific antibody designated as D4.

[0227] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide as well as polynucleotides that include additional coding and / or non-coding sequences. The polynucleotides disclosed in the present invention can be in the form of RNA or in the form of DNA. The DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. The single-stranded DNA can be the coding strand or the non-coding (antisense) strand. The polynucleotides disclosed in the present invention can be mRNA.

[0228] The present disclosure also provides variants of the polynucleotides described herein, wherein the variants have a nucleotide sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity with the polynucleotide sequence encoding at least one polypeptide chain of the anti-TNFa / IL23p19 dual-specificity antibody described herein. As used herein, the phrase "a polynucleotide having a nucleotide sequence having at least about 95% identity with a polynucleotide sequence" means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to 5 point mutations in every 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence having at least 95% identity with a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or some nucleotides of up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, singly scattered among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0229] The polynucleotide variants may contain alterations in the coding region, non-coding region, or both. In some embodiments, the polynucleotide variants contain alterations that result in silent substitutions, additions, or deletions without altering the nature or activity of the encoded polypeptide. In some embodiments, the polynucleotide variants contain silent substitutions (due to the degeneracy of the genetic code) that result in an unchanged amino acid sequence of the polypeptide. Polynucleotide variants may be generated for a variety of reasons, e.g., to optimize codon expression for a particular host (e.g., changing codons in a human mRNA to those preferred by a bacterial host such as Escherichia coli). In some embodiments, the polynucleotide variants contain at least one silent mutation in the non-coding or coding region of the sequence.

[0230] In some embodiments, polynucleotide variants are generated to regulate or alter the expression (or level of expression) of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to decrease the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant has increased expression of the encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variant has decreased expression of the encoded polypeptide compared to the parental polynucleotide sequence.

[0231] In some embodiments, the polynucleotide comprises a coding sequence of a polypeptide (e.g., an antibody) that is fused in the same reading frame with a polynucleotide that is expressed and secreted from a host cell with an auxiliary polypeptide (e.g., a leader sequence that functions as a secretion sequence for controlling polypeptide transport). The polypeptide may have a leader sequence that is cleaved by the host cell to form a "mature" form of the polypeptide.

[0232] In some embodiments, the polynucleotide comprises a coding sequence of a polypeptide (e.g., an antibody) that is fused in the same reading frame with a marker or tag sequence. For example, in some embodiments, the marker sequence is a hexahistidine tag (HIS-tag), which enables the polypeptide fused to the marker to be efficiently purified. In some embodiments, when using a mammalian host (e.g., COS-7 cells), the marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG TM tag. In some embodiments, a marker may be used in combination with other markers or tags.

[0233] In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure.

[0234] In some embodiments, provided herein are also vectors comprising the polynucleotides disclosed herein. As used herein, the term "vector" and its grammatical equivalents refer to a vehicle that can be introduced into a host cell for carrying genetic material (e.g., a polynucleotide sequence), in which it can replicate and / or be expressed. Suitable vectors to use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Additionally, the vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included provide, for example, antibiotic or toxin resistance, complementation of auxotrophies, or provide key nutrients not present in the medium. Expression control sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, etc., well known in the art. When co-expressing two or more polynucleotides, the two polynucleotides may be inserted, for example, into a single expression vector or different expression vectors. For single vector expression, the coding polynucleotides may be operably linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art can be used to confirm the introduction of the polynucleotide into the host cell. Those skilled in the art should understand to express the polynucleotide in an amount sufficient to produce the desired product, and should also understand that methods well known in the art can be used to optimize the expression level to obtain sufficient expression.

[0235] In some embodiments, the vectors provided herein can be expression vectors. In some embodiments, the vectors provided herein include a polynucleotide encoding at least one polypeptide chain of an anti-TNFa / IL23p19 bispecific antibody described herein. In some embodiments, recombinant expression vectors are provided herein, which can be used to amplify and express a polynucleotide encoding at least one polypeptide chain of an anti-TNFa / IL23p19 bispecific antibody described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes a synthetic or cDNA-derived DNA fragment encoding at least one polypeptide chain of an anti-TNFa / IL23p19 bispecific antibody described herein, operably linked to suitable transcriptional and / or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. In some embodiments, viral vectors are used. DNA regions are "operably linked" when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if its location enables translation. In some embodiments, structural elements designed for use in certain expression systems include a leader sequence that enables a host cell to secrete the translated protein extracellularly. In some embodiments, when a recombinant protein is expressed in the absence of a leader sequence or a transit sequence, the polypeptide may include an N-terminal methionine residue.

[0236] Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli (E. coli), including pCR1, pBR322, pMB9, and their derivatives, and broader host range plasmids, such as M13 and other filamentous single-stranded DNA phages. Other exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages, such as lambda phage or M13 phage, and animal viruses. Examples of classes of animal viruses useful as vectors include, without limitation, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and polyomaviruses (e.g., SV40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST for lentivirus-mediated gene transfer and expression in mammalian cells TM and pLenti6 / V5-DEST TMand pLenti6.2 / V5-GW / lacZ (Invitrogen). Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Exemplary transposon systems can be used, such as Sleeping Beauty and PiggyBac, which can stably integrate into the genome (e.g., Ivics et al., Cell, 91(4):501–510 (1997); et al., (2007) Nucleic Acids Research. 35(12):e87).

[0237] In some embodiments, the vector is an episomal vector or an extrachromosomally maintained vector. As used herein, the term "episomal" refers to a vector that is capable of replication but does not integrate into the host chromosomal DNA and is not gradually lost from dividing host cells, and also means that the vector replicates extrachromosomally or episomally. The vector is engineered to have a DNA replication origin or "ori" encoding from lymphotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically a sequence corresponding to the oriP of EBV, the replication origin of lymphotropic herpesvirus or gamma herpesvirus. In some embodiments, the lymphotropic herpesvirus can be Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), squirrel monkey herpesvirus (HS), or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Generally, the host cell contains a viral replication transactivator protein that activates replication.

[0238] "Expression control sequences", "control elements", or "regulatory sequences" present in the expression vector are those non-translated regions of the vector - replication origin, selection cassette, promoter, enhancer, translation initiation signal (ribosome binding site sequence or Kozak sequence) introns, polyadenylation sequence, 5' and 3' untranslated regions - which interact with host cell proteins for transcription and translation. The strength and specificity of these elements can vary. Any number of suitable transcriptional and translational elements can be used, including ubiquitous promoters and inducible promoters, depending on the vector system and host used.

[0239] Exemplary ubiquitous expression control sequences that can be used in the present invention include (but are not limited to) the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) promoter (e.g., early or late), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and the P11 promoter from vaccinia virus, the elongation factor 1-α (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa β, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), the ubiquitin C promoter (UBC), the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and the β-actin promoter.

[0240] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), the cumate-inducible gene switch (WO 2002 / 088346), the tetracycline-dependent regulatory system, etc. The bispecific antibodies described herein can be produced by any methods known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise indicated, the practice of the present invention uses conventional techniques in molecular biology, microbiology, gene analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature.See, for example, Maniatis et al. (1982) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (1987 and annual updates); CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley & Sons (1987 and annual updates); Gait (ed.) (1984) OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press; Eckstein (ed.) (1991) OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, IRL Press; Birren et al. (eds.) (1999) GENOME ANALYSIS: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) ANTIBODY ENGINEERING, 2nd Edition, Oxford University Press; Lo (ed.) (2006) ANTIBODY ENGINEERING: METHODS AND PROTOCOLS (METHODS IN MOLECULAR BIOLOGY); Vol. 248, Humana Press, Inc; each of the foregoing references is incorporated herein by reference in its entirety.

[0241] The present disclosure also provides cells comprising a polynucleotide disclosed herein encoding at least one polypeptide chain of an anti-TNFα / IL23p19 bispecific antibody described herein. In some embodiments, the cells provided herein comprise polynucleotides encoding LC1, HC1, LC2, and HC2 of an anti-TNFα / IL23p19 bispecific antibody disclosed herein having a CrossMab-KIH structure. In some embodiments, the cells provided herein comprise multiple polynucleotides encoding LC1, LC2, HC1, and HC2 of an anti-TNFα / IL23p19 bispecific antibody disclosed herein having a CrossMab-KIH structure, jointly.

[0242] In some embodiments, the cells provided herein comprise a polynucleotide encoding both LC and HC of an anti-TNFα / IL23p19 bispecific antibody disclosed herein having an IgG-scFv structure, DVID-Ig, or SMAB structure. In some embodiments, the cells provided herein comprise a first polynucleotide encoding LC and a second polynucleotide encoding HC of an anti-TNFα / IL23p19 bispecific antibody disclosed herein having an IgG-scFv structure, DVID-Ig, or SMAB structure.

[0243] Cells comprising a vector disclosed herein are also contemplated. In some embodiments, the present disclosure provides host cells comprising a vector containing a polynucleotide disclosed herein. In some embodiments, the host cells provided herein comprise a vector or multiple vectors jointly containing polynucleotides encoding polypeptide chains of an anti-TNFα / IL23p19 bispecific antibody described herein. In some embodiments, the host cells provided herein produce an anti-TNFα / IL23p19 bispecific antibody described herein.

[0244] Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived), L-929 (mouse fibroblast-derived), C127 (mouse mammary tumor-derived), 3T3 (mouse fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical carcinoma-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can contain non-transcribed elements such as origins of replication, suitable promoters and enhancers linked to the gene to be expressed and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' untranslated sequences such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art. 4. Production methods

[0245] The present disclosure also provides methods for generating the anti-TNFα / IL23p19 bispecific antibodies disclosed herein. In some embodiments, the bispecific antibodies disclosed herein are composed of more than one polypeptide chain, and the polypeptide chains can be produced separately or together. In some embodiments, the methods provided herein produce at least one polypeptide chain of the bispecific antibodies disclosed herein. In some embodiments, the methods provided herein produce all of the polypeptide chains of the bispecific antibodies disclosed herein.

[0246] The bispecific antibodies or polypeptides described herein can be generated and isolated using methods known in the art. The polypeptides can be synthesized wholly or partially by chemical means (see, e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, A.K., THERAPEUTIC PEPTIDES AND PROTEINS, FORMULATION, PROCESSING AND DELIVERY SYSTEMS (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be carried out using a variety of solid-phase techniques (see, e.g., Roberge, Science 269:202 (1995); Merrifield, Methods Enzymol. 289:3 (1997)) and, for example, automated synthesis can be achieved using an ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions. Peptides can also be synthesized using combinatorial methods. These synthetic residues and polypeptides can be synthesized using a variety of procedures and methods known in the art (see, e.g., Organic Syntheses Collective Volumes, Gilman et al. (eds.) John Wiley & Sons, Inc., NY). Modified peptides can be generated by chemical modification methods (see, e.g., Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994)). Methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis can also be used to effect peptide sequence alterations, derivatizations, substitutions, and modifications. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res. 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315 (1985)), restriction-selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London Ser A 317:415 (1986)), and other techniques can be implemented on cloned DNA to generate the peptide sequences, variants, fusions, and chimeras of the invention and their alterations, derivatives, substitutions, and modifications.

[0247] A variety of host-expression vector systems can be used to recombinantly express the dual-specific antibodies described herein or one or more of their polypeptide chains. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Cloning and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts, as well as methods for protein production, including antibody production, are well known in the art. These host-expression systems represent the means by which the coding sequences for the dual-specific antibodies described herein can be produced and subsequently purified, and also represent the cells that can express in situ the dual-specific antibodies described herein when transformed or transfected with the appropriate polynucleotide coding sequences. These include (but are not limited to) microorganisms transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequences for the compounds described herein, such as bacteria (e.g., Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis)); yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the sequences encoding the compounds described herein; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the sequences encoding the compounds described herein; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the sequences encoding the molecular compounds described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphocytes (see U.S. Patent No. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell) having a recombinant expression construct with a promoter derived from the mammalian cell genome (e.g., metallothionein promoter) or a promoter from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0248] In bacterial systems, a variety of expression vectors can be advantageously selected based on the intended use of the protein to be expressed. For example, when large amounts of such a protein are to be produced, for the production of the pharmaceutical compositions of the bispecific antibodies described herein, vectors that direct high-level expression of protein products that are easy to purify may be desired. These vectors include (but are not limited to) the Escherichia coli (E. coli) expression vector pUR278 (Ruther et al. (1983), EMBO J. 2:1791-1794); pIN vectors (Inouye et al. (1985), Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989), J. Biol. Chem. 24:5503-5509); etc. pGEX vectors can also be used to express polypeptides as fusion proteins with glutathione S-transferase (GST). Typically, these proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione agarose beads and then elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0249] Expression vectors useful for eukaryotic hosts include (for example) vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. In mammalian host cells, some virus-based expression systems can be used. Examples of suitable mammalian host cell lines include (but are not limited to) COS-7 (simian kidney-derived), L-929 (mouse fibroblast-derived), C127 (mouse mammary tumor-derived), 3T3 (mouse fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical carcinoma-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and their variants. Mammalian expression vectors can contain non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' untranslated sequences such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a robust method for producing correctly folded and biologically functional proteins. The baculovirus system for producing heterologous proteins in insect cells is well known to those skilled in the art. Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.

[0250] Alternatively, it is possible to choose to modulate the expression of the inserted sequence, or to alter and process the host cell line of the gene product in a desired specific manner. These modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for protein function. For example, in certain embodiments, the antibodies described herein can be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a polyprotein precursor), which requires proteolytic cleavage by natural or recombinant cellular mechanisms to form the individual polypeptides of the bispecific antibodies described herein. Thus, the present disclosure encompasses engineered nucleic acid sequences encoding polyprotein precursor molecules comprising polypeptides of bispecific antibodies described herein, which include coding sequences capable of directing post-translational cleavage of the polyprotein precursor. Post-translational cleavage of the polyprotein precursor results in the production of the polypeptides of the bispecific antibodies described herein. Post-translational cleavage of the precursor molecules of polypeptides comprising the compounds described herein can occur in vivo (i.e., within a host cell by natural or recombinant cellular systems / mechanisms, e.g., furin cleavage at appropriate sites) or can occur in vitro (e.g., the polypeptide chains are incubated in a composition comprising a protease or peptidase with known activity and / or in a composition comprising conditions or reagents known to facilitate the desired proteolysis). Purification and modification of recombinant proteins are well known in the art, such that the design of polyprotein precursors can include embodiments readily understood by one of ordinary skill in the art. Any known protease or peptidase known in the art can be used for the described modification of precursor molecules.

[0251] Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. A suitable cell line or host system can be chosen to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells having cellular mechanisms for the appropriate processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. These mammalian host cells include (but are not limited to) CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20, and T47D, CRL7030, and Hs578Bst.

[0252] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the compounds described herein can be engineered. Without using an expression vector containing a viral origin of replication, host cells can be transformed with DNA controlled by suitable expression control elements (e.g., promoters, enhancers, sequences, transcriptional terminators, polyadenylation sites, etc.) and selectable markers. After introduction of the foreign DNA, the engineered cells can be allowed to grow in enriched medium for 1-2 days and then transferred to selective medium. The selectable markers in the recombinant plasmid confer selection tolerance and allow the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can in turn be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the compounds described herein. These engineered cell lines can be particularly useful in the screening and evaluation of compounds that directly or indirectly interact with the compounds described herein.

[0253] A number of selection systems can be used, including (but not limited to) herpes simplex virus thymidine kinase (Wigler et al., (1977), Cell 11:223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al (1992) Bioessays 14:495-500), and adenine phosphoribosyltransferase (Lowy et al. (1980), Cell 22:817-823) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Additionally, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) PNAS, 77:3567-3570; O'Hare et al. (1981) PNAS, 78:1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al. (1981) PNAS, 78:2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tolstoshev (1993), Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993), Science 260:926-932; and Morgan et al. (1993), Ann. Rev. Biochem. 62:191-217) and hygro, which confers resistance to hygromycin (Santerre et al. (1984) Gene 30:147-156). Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds.), 1994, CURRENT PROTOCOLS IN HUMAN GENETICS, John Wiley & Sons, NY.

[0254] The expression level of the bispecific antibodies or their polypeptide chains described herein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Volume 3 (Academic Press, New York, 1987)). When the marker in the vector system described herein is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is related to the nucleotide sequence of the protein of interest, the production of the protein of interest will also be increased (Crouse et al. (1983) Mol. Cell. Biol. 3:257-266).

[0255] Host cells can be co-transfected with more than one expression vector, each encoding a polypeptide chain of the bispecific antibodies described herein. The vectors can contain the same selectable marker, which enables equal expression of all polypeptides. Alternatively, a single vector encoding two or more polypeptides can be used. The coding sequences of the polypeptides of the compounds described herein can comprise cDNA or genomic DNA.

[0256] Once the bispecific antibodies described herein or the polypeptides described herein have been recombinantly expressed, they can be purified by any method known in the art for the purification of polypeptides, multi-proteins or antibodies (e.g., similar to antibody purification protocols based on antigen selectivity), e.g., by chromatography (e.g., ion exchange chromatography, affinity chromatography, specifically by affinity for the specific antigen (optionally, after protein A selection when the compound contains an Fc domain (or a portion thereof)) and size exclusion chromatography), centrifugation, differential solubility or by any other standard technique for polypeptide or antibody purification.

[0257] The present invention provides methods for producing the anti-TNFα / IL23p19 bispecific antibodies described herein or the polypeptide chains of the bispecific antibodies described herein, said methods comprising obtaining the cells described herein and expressing the polynucleotides described herein in said cells. In some embodiments, the methods further comprise isolating and purifying the bispecific antibodies or polypeptide chains described herein.

[0258] The binding of the bispecific antibodies described herein to human TNFα and / or IL23p19 can be tested, for example, by standard ELISA. Briefly, microtiter plates are coated with purified antigen and then blocked with bovine serum albumin. Antibody dilutions are added to each well and incubated. The plates are washed and incubated with a second reagent conjugated to horseradish peroxidase (HRP) (e.g., for human antibodies, a goat-anti-human IgG Fc-specific polyclonal reagent). After washing, the plates can be developed and analyzed by spectrophotometer. For binding to cell lines expressing human TNFα and / or IL23p19 but not to control cell lines that do not express the target antigen, the antibodies can be further tested by flow cytometry. Briefly, antibody binding can be evaluated by incubating CHO cells expressing TNFα and / or IL23p19 with the bispecific antibodies provided herein. The cells can be washed and binding detected by anti-human IgG Ab. Flow cytometry analysis can be performed using FACS or a flow cytometer (Becton Dickinson, San Jose, CA).

[0259] The reactivity of the anti-TNFα / IL23p19 bispecific antibodies provided herein with the target antigen can be further tested by Western blot, and other methods known in the art for analyzing the binding affinity, cross-reactivity, and binding kinetics of the various anti-TNFα / IL23p19 bispecific antibodies described herein include, for example, biolayer interferometry (BLI) using, for example, the Gator system (Probe Life) or the Octet-96 system (Sartorius AG), or surface plasmon resonance (SPR) analysis using a BIACORE 2000 SPR instrument (Biacore AB, Uppsala, Sweden). TM 2000 SPR instrument (Biacore AB, Uppsala, Sweden). TM Surface plasmon resonance (SPR) analysis. 5. Pharmaceutical compositions

[0260] The present invention also provides a pharmaceutical composition comprising the anti-TNFα / IL23p19 bispecific antibody disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the disclosed bispecific antibody and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is useful in the treatment of inflammatory diseases or autoimmune diseases.

[0261] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a material that is suitable for pharmaceutical administration to an individual together with an active agent and that does not, in a harmful manner, cause an undesirable biological effect or interaction with any other component of the pharmaceutical composition. In some embodiments, the pharmaceutical compositions disclosed herein may also contain one or more buffer systems, preservatives, tonicity agents, chelating agents, stabilizers, and / or surfactants and different combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. Reference may be made to REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 19th Edition, 1995.

[0262] In some embodiments, the pharmaceutical compositions provided herein contain the anti-TNFa / IL23p19 bispecific antibody provided herein. The anti-TNFa / IL23p19 bispecific antibody may be present in a variety of concentrations. In some embodiments, the pharmaceutical compositions provided herein contain from 1 to 1000 mg / mL of the anti-TNFa / IL23p19 bispecific antibody provided herein. In some embodiments, the pharmaceutical composition contains from 10 to 500 mg / mL, from 10 to 400 mg / mL, from 10 to 300 mg / mL, from 10 to 200 mg / mL, from 10 to 100 mg / mL, from 20 to 100 mg / mL, or from 50 to 100 mg / mL of the anti-TNFa / IL23p19 bispecific antibody provided herein. In some embodiments, the pharmaceutical compositions provided herein contain about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 120 mg / mL, about 150 mg / mL, about 180 mg / mL, about 200 mg / mL, about 300 mg / mL, about 500 mg / mL, about 800 mg / mL, or about 1000 mg / mL of the anti-TNFa / IL23p19 bispecific antibody provided herein. Those skilled in the art can readily adjust the dosage; for example, a decrease in purity may require an increase in dosage.

[0263] Pharmaceutically acceptable carriers that can be used in the compositions provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Based on the route of administration, the active ingredient (i.e., the anti-TNFa / IL23p19 bispecific antibody) can be coated in the material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

[0264] Also provided herein are pharmaceutical compositions or formulations that improve the stability of anti-TNFa / IL23p19 bispecific antibodies to enable their long-term storage. In some embodiments, the pharmaceutical compositions or formulations disclosed herein comprise: (a) the anti-TNFa / IL23p19 bispecific antibodies disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a bulking agent; and / or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or longer. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4°C, 25°C, or 40°C.

[0265] Buffers useful in the pharmaceutical compositions or formulations disclosed herein can be weak acids or bases that are used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. Suitable buffers can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties can also be based on the pH of the formulation. Common buffers include (but are not limited to) histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) and an isotonicity agent and is potentially pH-adjusted with an acid or base known in the art. In certain embodiments, the buffer is L-histidine. In certain embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

[0266] Stabilizers are added to pharmaceutical products to stabilize the products. These agents can stabilize proteins in different ways. Common stabilizers include (but are not limited to) amino acids such as glycine, alanine, lysine, arginine or threonine, carbohydrates such as glucose, sucrose, trehalose, raffinose or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrin or dextran of any kind and molecular weight, or PEG. In some embodiments, the stabilizer is selected to maximize the stability of the antibody in the lyophilized formulation. In certain embodiments, the stabilizer is sucrose and / or arginine.

[0267] Bulking agents can be added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby facilitating accurate dosing and handling thereof. Common bulking agents include (but are not limited to) lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate or magnesium stearate.

[0268] Surfactants are amphiphilic substances having hydrophilic and hydrophobic groups. Surfactants can be anionic, cationic, zwitterionic or nonionic. Examples of nonionic surfactants include (but are not limited to) alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxides, polypropylene oxides, fatty alcohols such as cetyl alcohol or oleyl alcohol, coconut amide MEA, coconut amide DEA, polysorbates or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0269] In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but can also include colloids, dispersions, emulsions and multiphase materials. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0270] In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized and a solvent and / or diluent is added thereto by a physician or patient prior to use.

[0271] The pharmaceutical compositions disclosed herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0272] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0273] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures such as the above and by including various antibacterial and antifungal agents, for example, both parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the compositions. In addition, prolonged absorption of injectable drug forms can be effected by including agents that delay absorption such as aluminum monostearate and gelatin.

[0274] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and reagents for active pharmaceutical substances is known in the art. In some embodiments, provided herein are pharmaceutical compositions comprising the anti-TNFa / IL23p19 bispecific antibodies or cells provided herein, wherein the compositions are suitable for topical administration.

[0275] The pharmaceutical compositions or formulations must generally be sterile and stable under the conditions of production and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants. In most cases, the compositions may include isotonic agents in the compositions, for example, sugars, polyols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be caused by including agents that retard absorption, for example, monostearates and gelatin in the compositions.

[0276] Sterile injectable solutions can be prepared by incorporating the required amounts of the active compound in a suitable solvent having one or a combination of the ingredients listed above, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle which comprises a basic dispersion medium and the required other ingredients from those listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, some of the preparation methods are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any other desired ingredients from its previously sterile-filtered solution.

[0277] The amount of the active ingredient that can be combined with the carrier material in the pharmaceutical compositions or formulations disclosed herein can vary. In some embodiments, the amount of the active ingredient that can be combined with the carrier material is an amount that produces a therapeutic effect. Generally, on a hundred percent basis, the amount will be in the range of from about 0.01% to about 99% active ingredient, from about 0.1% to about 70%, or from about 1% to about 30% active ingredient in combination with a pharmaceutically acceptable carrier.

[0278] The pharmaceutical compositions disclosed herein can be prepared with a carrier that will protect the active ingredient from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate copolymer, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. A variety of methods for preparing these formulations are patent protected or generally known to those skilled in the art. See, for example, SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0279] The present invention also provides a kit for preparing a pharmaceutical composition having the anti-TNFα / IL23p19 dual-specific antibody disclosed herein. In some embodiments, the kit comprises the anti-TNFα / IL23p19 dual-specific antibody disclosed herein in one or more containers and a pharmaceutically acceptable carrier. In another embodiment, the kit can comprise the anti-TNFα / IL23p19 dual-specific antibody disclosed herein for administration to a subject. In a specific embodiment, the kit comprises instructions regarding the preparation and / or administration of the anti-TNFα / IL23p19 dual-specific antibody. 6. Methods and uses

[0280] The anti-TNFα / IL23p19 bispecific antibodies provided herein can be used in medicine. Also provided herein are methods of reducing TNFα and / or IL23p19-related autoimmunity or inflammation in a subject in need thereof, which comprise administering to the subject a therapeutically effective amount of the anti-TNFα / IL23p19 bispecific antibody disclosed herein. Also provided herein are methods of treating an autoimmune disease in a subject in need thereof, which comprise administering to the subject a therapeutically effective amount of the anti-TNFα / IL23p19 bispecific antibody disclosed herein. Also provided herein are methods of treating an inflammatory disease in a subject in need thereof, which comprise administering to the subject a therapeutically effective amount of the anti-TNFα / IL23p19 bispecific antibody disclosed herein. In some embodiments, the subject is a human. In some embodiments, the subject has an autoimmune disease. In some embodiments, the subject has an inflammatory disease. In some embodiments, the subject is at risk of developing an autoimmune disease. In some embodiments, the subject is at risk of developing an inflammatory disease.

[0281] Provided herein is the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein as a medicament. Also provided herein is the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein in the treatment of autoimmune diseases. Also provided herein is the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein in the treatment of inflammatory diseases. Provided herein is the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein for the preparation of a medicament for the treatment of autoimmune diseases. Also provided herein is the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein for the preparation of a medicament for the treatment of inflammatory diseases.

[0282] As used herein, the term "treat" and its grammatical equivalents in relation to a disease or condition or a subject having a disease or condition denote an action of inhibiting, eliminating, reducing, and / or ameliorating the symptoms, severity of symptoms, and / or frequency of symptoms associated with the disease or disorder being treated.

[0283] As used herein, the term "administer" and its grammatical equivalents refer to the act of delivering or causing the delivery of a therapeutic agent or pharmaceutical composition to the body of a subject by the methods described herein or otherwise known in the art. The therapeutic agent can be a compound, polypeptide, antibody, cell, or population of cells. Administering a therapeutic agent or pharmaceutical composition includes prescribing a therapeutic agent or pharmaceutical composition to be delivered to the body of a subject. Exemplary forms of administration include oral dosage forms such as tablets, capsules, syrups, suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP) injectable dosage forms; transdermal dosage forms including creams, gels, powders, or patches; oral dosage forms; inhaled powders, sprays, suspensions, and rectal suppositories.

[0284] As used herein, the terms "effective amount," "therapeutically effective amount," and their grammatical equivalents mean an amount of a reagent that, when administered to the subject, alone or as part of a pharmaceutical composition and in a single dose or as part of a series of doses, is administered to the subject in an amount capable of having any detectable positive effect on any symptom, aspect, or feature of a disease, disorder, or condition. The therapeutically effective amount can be determined by measuring the relevant physiological effects. The exact amount required varies from subject to subject based on factors such as the subject's age, weight, and general condition, the severity of the condition to be treated, and the judgment of the clinician, among others. One of ordinary skill in the art can determine the appropriate "effective amount" in any individual case using routine experimentation.

[0285] As used herein, the term "subject" refers to any animal (e.g., a mammal), including (but not limited to) a human, non-human primate, dog, cat, rodent, etc., that will be the recipient of a particular treatment. Mammals include (but are not limited to) livestock, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment can be a human subject suffering from a disease, at risk of developing a disease, or suspected of having a disease. A subject suffering from a disease can be identified by routine medical examinations, e.g., physical examinations, laboratory tests, organ function tests, CT scans, or ultrasounds. A subject suspected of having any such disease can exhibit one or more symptoms of the disease. The signs and symptoms of diseases, e.g., autoimmune and inflammatory diseases, are well known to those of skill in the art. A subject at risk of developing a disease can be a subject having one or more risk factors for the disease. The subject can be a human. The subject can be suffering from a particular disease or condition.

[0286] Non-limiting examples of autoimmune diseases include rheumatoid arthritis, psoriasis, type 1 diabetes, systemic lupus erythematosus, transplant rejection, autoimmune thyroid disease (Hashimoto's disease), sarcoidosis, scleroderma, granulomatosis with polyangiitis, Crohn's disease, ulcerative colitis, Sjogren's disease, ankylosing spondylitis, psoriatic arthritis, polymyositis, dermatomyositis, polyarteritis nodosa, immune-mediated bullous skin diseases, Behcet's syndrome, multiple sclerosis, systemic sclerosis, hidradenitis suppurativa, palmoplantar pustulosis, pityriasis rubra pilaris, atopic dermatitis, juvenile idiopathic arthritis, Goodpasture's disease, or immune-mediated glomerulonephritis.

[0287] Non-limiting examples of inflammatory diseases include rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos' disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, inflammatory skeletal muscle degeneration, insulin-dependent diabetes (type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, cirrhosis of the liver of Charcot, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and orbital necrotizing granulomatosis. In some embodiments, the autoimmune or inflammatory disease is Crohn's disease, ankylosing spondylitis, or psoriatic arthritis.

[0288] In some embodiments, the bispecific antibodies and pharmaceutical compositions provided herein can be used to treat autoimmune or inflammatory diseases, which are plaque psoriasis, hidradenitis suppurativa, palmoplantar pustulosis, pityriasis rubra pilaris, atopic dermatitis, systemic sclerosis, Takayasu arteritis, giant cell arteritis, uveitis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, intestinal Behçet's disease, or inflammatory myopathy. In some embodiments, the autoimmune or inflammatory disease is plaque psoriasis. In some embodiments, the autoimmune or inflammatory disease is psoriatic arthritis. In some embodiments, the autoimmune or inflammatory disease is Crohn's disease. In some embodiments, the autoimmune or inflammatory disease is ulcerative colitis. In some embodiments, the autoimmune or inflammatory disease is palmoplantar pustulosis.

[0289] In the methods and uses disclosed herein, a therapeutically effective amount of the anti-TNFα / IL23p19 bispecific antibody or pharmaceutical composition disclosed herein is administered to a subject who can benefit from reduced autoimmunity. The subject can have undesired, unrestricted, or excessive autoimmune activation. The subject can be at risk of developing undesired, unrestricted, or excessive autoimmune activation. The actual dosage level of the active ingredient (i.e., the anti-TNFα / IL23p19 bispecific antibody disclosed herein) in the pharmaceutical composition described herein can be varied to obtain an amount of the active ingredient that is effective for achieving the desired therapeutic response for a particular patient, composition, and mode of administration but is non-toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition described herein, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0290] The anti-TNFα / IL23p19 bispecific antibody disclosed herein can be administered as a sustained-release formulation, in which case frequent administration is not required. The dosage and frequency are varied based on the half-life of the anti-TNFα / IL23p19 bispecific antibody in the patient. In therapeutic applications, relatively high doses at relatively short intervals are sometimes required until disease progression is reduced or terminated and until the patient shows partial or complete improvement of the disease symptoms.

[0291] The anti-TNFα / IL23p19 bispecific antibody or pharmaceutical composition provided herein can be administered to a subject by any method known in the art, including (but not limited to) pleural administration, intravenous administration, subcutaneous administration, intranodular administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral administration routes, e.g., by injection or infusion, or directly into the thymus. As used herein, the phrase "parenteral administration" refers to a form of administration other than enteral and topical administration, which is generally by injection and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some embodiments, subcutaneous administration is employed. In some embodiments, intravenous administration is employed. In some embodiments, oral administration is employed. In one embodiment, the antibody or antigen-binding fragment provided herein can be delivered locally. In another embodiment, the antibody or antigen-binding fragment provided herein can be administered systemically.

[0292] The anti-TNFα / IL23p19 bispecific antibody or pharmaceutical composition provided herein can be administered by a medical device known in the art. For example, in some embodiments, a needleless subcutaneous injection device can be used, such as the devices disclosed in the following: U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use in the present description include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering agents through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses a permeable drug delivery system having multiple compartments; and U.S. Patent No. 4,475,196, which discloses a permeable drug delivery system. These patents are incorporated herein by reference. Numerous other such implants, delivery systems, and modules are known to those skilled in the art.

[0293] In some embodiments, the anti-TNFα / IL23p19 bispecific antibodies or pharmaceutical compositions provided herein can be administered in combination with other therapies. The other therapies can be administered before, simultaneously with, or after the administration of the bispecific antibodies or pharmaceutical compositions described herein. The combination administration can include co-administration in a single pharmaceutical formulation or using different formulations, or sequential administration in either order but generally within a time period such that all of the active agents can exert their biological activities simultaneously. Those skilled in the art can readily determine an appropriate regimen for the combination administration of the pharmaceutical compositions described herein and other therapies, including the timing and dosage of the other reagents used in the combination therapy. 7. Experiments

[0294] Unless otherwise indicated, the following examples are provided for illustrative purposes only and are not intended to be limiting. Thus, the present invention should in no way be considered limited to the following examples, but rather should be considered to cover any and all modifications that become apparent as a result of the teachings provided herein.

[0295] Briefly, the results of the studies described below confirm that the bispecific antibodies bind to both human TNFα and human IL23p19 with high affinity and effectively neutralize the biological activities of human TNFα and human IL23p19 in vitro and in vivo.

[0296] The reference antibodies used in these studies included adalimumab (light and heavy chains: SEQ ID NOs: 3 and 4, respectively), golimumab (light and heavy chains: SEQ ID NOs: 7 and 8, respectively), Ozoralizumab (SEQ ID NO: 9), guselkumab (light and heavy chains: SEQ ID NOs: 12 and 13, respectively), and tildrakizumab (light and heavy chains: SEQ ID NOs: 16 and 17, respectively). 7.1 Example 1: Expression and purification of bispecific antibodies

[0297] Methods: As shown below, the exemplified bispecific antibody was expressed and purified. ExpiCHO-S cells growing actively were inoculated into a serum-free expression medium and cultivated in an incubator at 37°C and 8% CO2. Cell contamination was carefully checked and the cell density was counted. The plasmid was mixed with a transfection reagent and incubated at room temperature for 2 min. The mixture was added to Expi CHO-s cells and incubated. At 18 - 22 h post transfection, enhancer and feed were added to the cells. The cells were centrifuged to collect the supernatant. The supernatant was filtered through a 0.22 μm microfiltration membrane and loaded onto a purification column. The antibody eluate was transferred to a dialysis bag and dialyzed against PBS. The bispecific antibody sample was stored at 4°C for testing; the remainder was frozen in liquid nitrogen and stored at -70°C. 7.2 Example 2: Binding Affinity of Bispecific Antibody to TNFα and IL23p19

[0298] Methods : The running buffer (HBS-EP+) was prepared by diluting 1 volume of 10× stock solution with 9 volumes of degassed, filtered MilliQ water. The regeneration buffer (10 mM glycine) was prepared by dissolving glycine in MilliQ water and adjusting the pH to 1.5 - 1.7. The assay was performed at 25°C. The antibody was injected as a capture on a Series S Sensor Chip Protein A. As the binding phase, recombinant antigen (TNFα or IL23p19) was injected at different concentrations on the surfaces of flow cells 1 and 2, and then the running buffer was injected as the dissociation phase. Affinity data were obtained by the analysis software.

[0299] Results and conclusions : As Figure 2 shown, the exemplified bispecific antibody disclosed herein binds to both human TNFα and human IL23p19 with high affinity comparable to those of the reference antibodies. 7.3 Example 3: Simultaneous Binding of TNFα and IL-23p19

[0300] Methods : First, the bispecific antibody was captured on a Protein A sensor chip, and then 100 nM human IL-23 was injected for 120 s to saturate the IL-23 binding sites of the bispecific antibody. Then, as the binding phase, human TNFα was injected at a series of concentrations, and subsequently, the running buffer was injected as the dissociation phase. Only one orientation was applied. Affinity data were obtained by the analysis software.

[0301] Results and conclusions : As Figure 3As shown, the exemplary bispecific antibodies disclosed herein bind simultaneously to human TNFα and human IL23p19. Unexpectedly, the TNFα binding affinity of the bispecific antibodies pre-bound to IL23p19 is comparable to their affinity for TNFα as a single antigen as measured in Example 2, indicating that by separately and simultaneously targeting these two antigens, dual therapeutic purposes can be achieved with the exemplified bispecific antibodies, and these antibodies can be used as a safer and more effective therapeutic option than antibodies targeting a single antigen. 7.4 Example 4: Inhibition of TNFα-induced NF-κB luciferase signal in TNFR reporter cell line

[0302] Methods : The inhibitory activity of the exemplary bispecific antibodies provided herein against the soluble TNFα signal transduction pathway was measured by a TNFα neutralization assay using a luciferase reporter cell line. The cell line was engineered to contain an NF-κB response element upstream of the luciferase reporter gene. The signal cascade that activates NF-κB, mediated by TNFα binding to the cell surface TNFR, in turn promotes the expression of the luciferase reporter gene. As shown, serially diluted samples of the exemplary bispecific antibodies were pre-incubated with TNFα in a 96-well plate at ambient temperature. After incubation, the cells were transferred to the wells in the assay plate and incubated. The TNFα neutralization potency was determined by luciferase signal.

[0303] Results and conclusions : As Figure 4A , Figure 4B and Figure 4C shown, the exemplary bispecific antibodies disclosed herein effectively inhibit human TNFα-induced NF-κB signal transduction with high potency. As shown, the inhibitory potency of the exemplary bispecific antibodies provided herein is comparable to that observed with the reference antibody. 7.5 Example 5: Inhibition of IL-23-induced STAT3 phosphorylation luciferase signal in IL-23R reporter cell line

[0304] Methods : The inhibitory activity of the exemplary bispecific antibodies provided herein against the IL-23 signal transduction pathway was measured by an IL-23 neutralization assay using a luciferase reporter cell line. Incubation of this reporter cell line with human IL-23 results in phosphorylation of STAT3 mediated by IL-23R / JAK kinases, and this phosphorylation was measured using a commercially available luciferase reporter gene assay reagent. As shown, serially diluted samples of the exemplary bispecific antibodies were pre-incubated with IL-23 in a 96-well plate at ambient temperature. After incubation, the cells were transferred to the wells in the assay plate and incubated. The IL-23 neutralization potency was determined by luciferase signal.

[0305] Results and conclusions : As Figure 5A , Figure 5B and Figure 5C shown, the exemplary bispecific antibodies disclosed herein effectively inhibit human IL-23-induced STAT3 phosphorylation signal transduction with high potency. As shown, the inhibitory potency of the exemplary bispecific antibodies provided herein is comparable to that observed with a reference antibody. 7.6 Example 6: Inhibition of apoptosis of TNFα-induced U-937 cells

[0306] Methods : U-937 is a promonocytic human myeloid leukemia cell line that naturally expresses TNF receptors. TNFα induces caspase-dependent apoptosis of cells in U-937 cells. Inhibition of TNFα-induced apoptosis of cells was determined by measuring the activity of caspase 3 / 7. Serial dilutions of the bispecific antibody were incubated with U-937 cells for 48 h. After incubation, the luminescence signal was measured by the 3 / 7 assay system.

[0307] Results and conclusions : As Figure 6 shown, the exemplary bispecific antibodies disclosed herein effectively inhibit apoptosis of human TNFα-induced U937 cells. The inhibitory potency of the exemplary bispecific antibodies provided herein is comparable to that observed with a reference antibody. 7.7 Example 7: Inhibition of TNFα-induced cytotoxicity of cells in L929

[0308] Methods : L929 is a murine fibroblast cell line that naturally expresses TNF receptors. TNFα induces L929 cell death due to the excessive formation of reactive oxygen intermediates. Inhibition of the cytotoxicity of cells by the bispecific antibody was determined by measuring cell viability. Serial dilutions of the bispecific antibody were pre-incubated with TNF-α and added to L929 cells. The mixture was incubated at 37 °C / 5% CO2. After incubation, the cytotoxicity level of the cells was determined by measuring the luminescence signal.

[0309] Results and conclusions : As Figure 7 shown, the exemplary bispecific antibodies disclosed herein effectively inhibit the cytotoxicity of human TNFα-induced L929 cells. The inhibitory potency of the exemplary bispecific antibodies provided herein is comparable to that observed with a reference antibody. 7.8 Example 8: Inhibition of TNFα and IL-23 combination-induced IL-17 cytokine production in human PBMC

[0310] Methods : It is known that TNFα enhances the secretion of IL-17 from Th17 cells. Human PBMCs were treated with a cytokine mixture containing TNFα and IL-23 that had been pre-incubated with titrated bispecific antibodies. Cell supernatants were collected and the concentration of IL-17 was analyzed using a commercial ELISA kit.

[0311] Results and conclusions : As Figure 8 shown, the exemplary bispecific antibodies disclosed herein effectively inhibit the production of human IL-17 cytokine induced by the combination of TNFα and IL-23 in human PBMCs. The inhibitory potency of the exemplary bispecific antibodies provided herein is superior to that observed with reference antibodies. 7.9 Example 9: Inhibition of human TNFα-induced mIL-6 production in mice

[0312] Methods : Mice were injected with the bispecific antibodies provided herein and then challenged with human TNFα to induce in vivo production of IL-6. After challenge, longitudinal serum was collected from whole blood and the IL-6 levels in the mice were analyzed using a commercial ELISA assay kit.

[0313] Results and conclusions : As Figure 9 shown, the exemplary bispecific antibodies disclosed herein significantly inhibit human TNFα-induced in vivo production of IL-6 in mice. 7.10 Example 10: Inhibition of human IL-23-induced ear hyperplasia in mice

[0314] Methods : Mice were injected with bispecific antibodies and then challenged intradermally with human IL-23 to induce ear hyperplasia. Ear thickness, ear tissue cytokines, and ear tissue pathology scores were evaluated.

[0315] Results and conclusions : As Figure 10 shown, the exemplary bispecific antibodies disclosed herein significantly improve ear thickness, clinical PASI scores, and histopathology induced by human IL-23 in a mouse ear hyperplasia model. The inhibitory potency of the bispecific antibodies provided herein is comparable to that observed with reference antibodies.

[0316] Accordingly, compared to monospecific antibodies targeting TNFα or anti-IL23 antibodies or combinations thereof, the bispecific antibodies provided herein combine additive and / or synergistic therapeutic potency, comparable safety, reduced cost, and improved convenience, safety, and patient compliance as a single reagent. Reference to the electronically submitted sequence listing

[0317] This application for invention incorporates by reference a Sequence Listing entitled "[P22406692C].SEQ.XML", which was created on September 18, 2022 and is 142,259 bytes in size.

Claims

1. A bispecific antibody that specifically binds to human TNFα (TNFα) and binds to the P19 subunit of human IL-23 (IL23p19), comprising: (1) A first light chain (LC1) comprising a first light chain variable domain (VL1) and a first heavy chain constant domain 1 (CH1); (2) A first heavy chain (HC1) comprising a first heavy chain variable domain (VH1), a first light chain constant region (CL), and a stamen-Fc region; (3) A second light chain (LC2) comprising a second light chain variable domain (VL2) and a second CL region; and (4) A second heavy chain (HC2) comprising a second heavy chain variable domain (VH2), a second CH1 domain, and a mortise-Fc region; wherein (i) The VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively; and (ii) The stamen-Fc region is a variant of the human IgG Fc region having a T366W substitution (according to EU index numbering); and the mortise-Fc region is a variant of the human IgG Fc region having a Y407V substitution (according to EU index numbering).

2. The bispecific antibody according to claim 1, wherein (1) The first CL region is kappa CL (Cκ; SEQ ID NO:68) or lambda CL (Cλ, SEQ ID NO:69); and the second CL region is Cκ (SEQ ID NO:68) or Cλ (SEQ ID NO:69); (2) Both the first CH1 domain and the second CH1 domain are human IgG1 CH1 domains (SEQ ID NO:61); or (3) The stamen-Fc region has the amino acid sequence shown in SEQ ID NO:66; and the mortise-Fc region has the amino acid sequence shown in SEQ ID NO:67; or any combination of (1)-(3).

3. The bispecific antibody according to claim 1 or 2, wherein the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19.

4. The bispecific antibody according to claim 3, wherein VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO:1 and 2, respectively; or (2) the amino acid sequences shown by SEQ ID NO:5 and 6, respectively; or wherein VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO:10 and 11, respectively; or (2) the amino acid sequences shown by SEQ ID NO:14 and 15, respectively.

5. The bispecific antibody according to claim 3, wherein VL1, VH1, VL2, and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 1, 2, 10, and 11 respectively; (2) the amino acid sequences shown by SEQ ID NO: 1, 2, 14, and 15 respectively; (3) the amino acid sequences shown by SEQ ID NO: 5, 6, 10, and 11 respectively; or (4) the amino acid sequences shown by SEQ ID NO: 5, 6, 14, and 15 respectively.

6. The bispecific antibody according to claim 3, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 24, 25, 12, and 26 respectively; (2) SEQ ID NO: 27, 28, 12, and 26 respectively; (3) SEQ ID NO: 24, 25, 16, and 29 respectively; or (4) SEQ ID NO: 27, 28, 16, and 29 respectively.

7. The bispecific antibody according to claim 1 or 2, wherein the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα.

8. The bispecific antibody according to claim 7, wherein VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 14 and 15 respectively; or wherein VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 5 and 6 respectively.

9. The bispecific antibody according to claim 7, wherein VL1, VH1, VL2, and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 10, 11, 1, and 2 respectively; (2) the amino acid sequences shown by SEQ ID NO: 10, 11, 5, and 6 respectively; (3) the amino acid sequences shown by SEQ ID NO: 14, 15, 1, and 2 respectively; or (4) the amino acid sequences shown by SEQ ID NO: 14, 15, 5, and 6 respectively.

10. The bispecific antibody according to claim 7, wherein LC1, HC1, LC2, and HC2 have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 18, 19, 3, and 20 respectively; (2) SEQ ID NO: 21, 22, 3, and 20 respectively; (3) SEQ ID NO: 18, 19, 7, and 23 respectively; or (4) SEQ ID NO: 21, 22, 7, and 23 respectively.

11. A bispecific antibody that specifically binds to human TNFα and binds to human IL23p19, comprising: (1) a light chain (LC) comprising a first light chain variable domain (VL1) and a CL region; and (2) A heavy chain (HC) comprising a first heavy chain variable domain (VH1), a heavy chain constant region (CH), a second light chain variable domain (VL2); and a second heavy chain variable domain (VH2); wherein the VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively.

12. The bispecific antibody according to claim 11, wherein (1) the CL region is Cκ (SEQ ID NO:68) or Cλ (SEQ ID NO:69); or (2) the CH region is a human IgG1 CH region (SEQ ID NO:58); or both (1) and (2).

13. The bispecific antibody according to claim 11 or 12, wherein the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19.

14. The bispecific antibody according to claim 13, wherein VL1 and VH1 have (1) amino acid sequences shown by SEQ ID NO:1 and 2, respectively; or (2) amino acid sequences shown by SEQ ID NO:5 and 6, respectively; or wherein VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO:10 and 11, respectively; or (2) amino acid sequences shown by SEQ ID NO:14 and 15, respectively; or (3) amino acid sequences shown by SEQ ID NO:93 and 94, respectively.

15. The bispecific antibody according to claim 13, wherein VL1, VH1, VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO:1, 2, 10 and 11, respectively; (2) amino acid sequences shown by SEQ ID NO:1, 2, 14 and 15, respectively; (3) amino acid sequences shown by SEQ ID NO:1, 2, 93 and 94, respectively; (4) amino acid sequences shown by SEQ ID NO:5, 6, 10 and 11, respectively; or (5) amino acid sequences shown by SEQ ID NO:5, 6, 14 and 15, respectively; (6) amino acid sequences shown by SEQ ID NO:5, 6, 93 and 94, respectively.

16. The bispecific antibody according to claim 13, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with (1) amino acid sequences shown by SEQ ID NO:3 and 30, respectively; (2) amino acid sequences shown by SEQ ID NO:3 and 31, respectively; (3) amino acid sequences shown by SEQ ID NO:7 and 32, respectively; (4) amino acid sequences shown by SEQ ID NO:7 and 33, respectively; or (5) amino acid sequences shown by SEQ ID NO:7 and 92, respectively.

17. The bispecific antibody according to claim 11 or 12, wherein the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα.

18. The bispecific antibody according to claim 17, wherein VL1 and VH1 have (1) amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) amino acid sequences shown by SEQ ID NO: 14 and 15 respectively; or wherein VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) amino acid sequences shown by SEQ ID NO: 5 and 6 respectively.

19. The bispecific antibody according to claim 17, wherein VL1, VH1, VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 10, 11, 1 and 2 respectively; (2) amino acid sequences shown by SEQ ID NO: 10, 11, 5 and 6 respectively; (3) amino acid sequences shown by SEQ ID NO: 14, 15, 1 and 2 respectively; or (4) amino acid sequences shown by SEQ ID NO: 14, 15, 5 and 6 respectively.

20. The bispecific antibody according to claim 17, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with (1) amino acid sequences shown by SEQ ID NO: 12 and 34 respectively; (2) amino acid sequences shown by SEQ ID NO: 12 and 35 respectively; (3) amino acid sequences shown by SEQ ID NO: 16 and 36 respectively; or (4) amino acid sequences shown by SEQ ID NO: 16 and 37 respectively.

21. A bispecific antibody that specifically binds to human TNFα and binds to human IL23p19, comprising: (1) a light chain (LC) that comprises a first light chain variable domain (VL1), a second light chain variable domain (VL2) and a CL region; and (2) a heavy chain (HC) that comprises a first heavy chain variable domain (VH1), a second heavy chain variable domain (VH2) and a CH region; wherein the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19 respectively, or specifically bind to human IL23p19 and human TNFα respectively.

22. The bispecific antibody according to claim 21, wherein (1) the CL region is Cκ (SEQ ID NO: 68) or Cλ (SEQ ID NO: 69); or (2) the CH region is a human IgG1 CH region (SEQ ID NO: 58); or both (1) and (2).

23. The bispecific antibody according to claim 21 or 22, wherein the VL1 / VH1 pair specifically binds to human TNFα and the VL2 / VH2 pair specifically binds to human IL23p19.

24. The bispecific antibody according to claim 23, wherein VL1 and VH1 have (1) amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) amino acid sequences shown by SEQ ID NO: 5 and 6 respectively; or wherein VL2 and VH2 have (1) amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) amino acid sequences shown by SEQ ID NO: 14 and 15 respectively.

25. The bispecific antibody according to claim 23, wherein VL1, VH1, VL2, and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 1, 2, 10, and 11 respectively; (2) the amino acid sequences shown by SEQ ID NO: 1, 2, 14, and 15 respectively; (3) the amino acid sequences shown by SEQ ID NO: 5, 6, 10, and 11 respectively; or (4) the amino acid sequences shown by SEQ ID NO: 5, 6, 14, and 15 respectively.

26. The bispecific antibody according to claim 23, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 46 and 47 respectively; (2) SEQ ID NO: 48 and 49 respectively; (3) SEQ ID NO: 50 and 51 respectively; or (4) SEQ ID NO: 52 and 53 respectively.

27. The bispecific antibody according to claim 21 or 22, wherein the VL1 / VH1 pair specifically binds to human IL23p19 and the VL2 / VH2 pair specifically binds to human TNFα.

28. The bispecific antibody according to claim 27, wherein VL1 and VH1 have (1) the amino acid sequences shown by SEQ ID NO: 10 and 11 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 14 and 15 respectively; or wherein VL2 and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 1 and 2 respectively; or (2) the amino acid sequences shown by SEQ ID NO: 5 and 6 respectively.

29. The bispecific antibody according to claim 27, wherein VL1, VH1, VL2, and VH2 have (1) the amino acid sequences shown by SEQ ID NO: 10, 11, 1, and 2 respectively; (2) the amino acid sequences shown by SEQ ID NO: 10, 11, 5, and 6 respectively; (3) the amino acid sequences shown by SEQ ID NO: 14, 15, 1, and 2 respectively; or (4) the amino acid sequences shown by SEQ ID NO: 14, 15, 5, and 6 respectively.

30. The bispecific antibody according to claim 27, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown by (1) SEQ ID NO: 38 and 39 respectively; (1) SEQ ID NO: 40 and 41 respectively; (3) SEQ ID NO: 42 and 43 respectively; or (4) SEQ ID NO: 44 and 45 respectively.

31. A bispecific antibody that specifically binds to human TNFα and binds to human IL23p19, comprising: (1) a light chain (LC) comprising a light chain variable domain (VL) and a CL region; and (2) a heavy chain (HC) comprising a heavy chain variable domain (VH) and a CH region; wherein (1) The VL / VH pair specifically binds to human IL23p19; and (2) the HC further comprises a single-chain variable domain antibody (VHH) that specifically binds to human TNFα.

32. The bispecific antibody according to claim 31, wherein (1) the CL region is Cκ (SEQ ID NO:68) or Cλ (SEQ ID NO:69); or (2) the CH region is a human IgG1 CH region (SEQ ID NO:58); or both (1) and (2).

33. The bispecific antibody according to claim 31 or 32, wherein VL and VH have (1) amino acid sequences shown by SEQ ID NO:10 and 11 respectively; or (2) amino acid sequences shown by SEQ ID NO:14 and 15 respectively.

34. The bispecific antibody according to any one of claims 31 to 33, wherein VHH has the amino acid sequence shown by SEQ ID NO:

9.

35. The bispecific antibody according to any one of claims 31 to 34, wherein VHH is linked to the N-terminus of VH.

36. The bispecific antibody according to claim 35, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with (1) amino acid sequences shown by SEQ ID NO:12 and 54 respectively; or (2) amino acid sequences shown by SEQ ID NO:16 and 56 respectively.

37. The bispecific antibody according to any one of claims 31 to 34, wherein VHH is linked to the C-terminus of the Fc domain.

38. The bispecific antibody according to claim 37, wherein LC and HC have at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with (1) amino acid sequences shown by SEQ ID NO:12 and 55 respectively; or (2) amino acid sequences shown by SEQ ID NO:16 and 57 respectively.

39. A polynucleotide encoding LC1, LC2, HC1, HC2 or any combination thereof of the bispecific antibody according to any one of claims 1 to 10.

40. A polynucleotide encoding LC, HC or both LC and HC of the bispecific antibody according to any one of claims 11 to 38.

41. A vector comprising the polynucleotide according to claim 50 or 51.

42. A cell comprising (a) the polynucleotide according to claim 50 encoding LC1, LC2, HC1 and HC2, or (b) multiple polynucleotides according to claim 50 that jointly encode LC1, LC2, HC1 and HC2.

43. A cell comprising (a) the polynucleotide according to embodiment 51 encoding both LC and HC, or (b) a first polynucleotide according to embodiment 51 encoding LC and a second polynucleotide according to embodiment 51 encoding HC.

44. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, and / or a cell according to claim 42 or 43, and optionally a pharmaceutically acceptable carrier.

45. A method of reducing TNFα and / or IL23p19-related autoimmunity or inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, a cell according to claim 42 or 43, and / or a pharmaceutical composition according to claim 44.

46. The method according to claim 45, wherein the subject has an autoimmune disease or an inflammatory disease.

47. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, a cell according to claim 42 or 43, and / or a pharmaceutical composition according to claim 44.

48. A method of treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, a cell according to claim 42 or 43, and / or a pharmaceutical composition according to claim 44.

49. The method according to any one of claims 45 to 48, wherein the subject is a human.

50. Use of a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, or a cell according to claim 42 or 43 as a medicament.

51. Use of a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, a cell according to claim 42 or 43, or a pharmaceutical composition according to claim 44 in the treatment of an autoimmune disease.

52. Use of a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, a cell according to claim 42 or 43, or a pharmaceutical composition according to claim 44 in the treatment of an inflammatory disease.

53. Use of a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, or a cell according to claim 42 or 43 for the preparation of a medicament for the treatment of an autoimmune disease.

54. Use of a bispecific antibody according to any one of claims 1 to 38, a polynucleotide according to claim 39 or 40, or a cell according to claim 42 or 43 for the preparation of a medicament for the treatment of an inflammatory disease.

55. A method for generating a bispecific antibody that specifically binds to human TNFα and binds to human IL23p19 by expressing the polynucleotide or the plurality of polynucleotides in the cell according to claim 42 or 43.

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