Antibodies targeting TNFα and IL-23, and their use

Abstract

This disclosure discloses bispecific antibodies that target both human TNFα and the P19 subunit of human IL-23. This disclosure further provides a polynucleotide encoding the bispecific antibody, a pharmaceutical composition comprising the bispecific antibody, and a method for producing the bispecific antibody. Furthermore, this disclosure discloses the medical uses of the bispecific antibody described herein.

Description

Detailed description of the invention

[0001] [Technical field] This disclosure relates to molecular biology and immunology. This specification provides bispecific antibodies that target both human TNFα (TNFα) and the P19 subunit of human IL-23 (IL23p19), as well as their use in the treatment of autoimmune and inflammatory diseases.

[0002] [Background technology] Autoimmune and inflammatory diseases arise when the body produces an immune response against its own tissues, often becoming chronic and potentially debilitating or life-threatening. TNFα is an effective inducer of inflammatory responses, and interleukin-23 (IL-23) is an upstream regulator in tissue inflammation. Both TNFα and IL-23 are clinically validated targets. However, there remains a significant unmet need for novel therapeutic options for patients who have not responded adequately to existing therapies targeting either TNFα or IL-23. The bispecific antibodies targeting both human TNFα and human IL-23p19, as well as related methods, provided herein, address these needs and offer relative advantages.

[0003] [Overview of the prefecture] This specification provides bispecific antibodies that specifically bind to human TNFα (TNFα) and the P19 subunit (IL23p19) of human IL-23, as well as related pharmaceutical compositions, polynucleotides, vectors, and cells. This specification also provides methods for producing these bispecific antibodies and methods for using these bispecific antibodies. Several exemplary embodiments are provided below.

[0004] Embodiment 1: (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 human IgG having a T366W substitution (numbered by EU index). This is a bispecific antibody that is an Fc region variant, with the Hole-Fc region being a variant of the human IgG Fc region having a Y407V substitution (numbered by the EU index), and specifically binds to human TNFα and human IL23p19.

[0005] Embodiment 2: The bispecific antibody according to Embodiment 1, wherein (1) the first CL region is κCL (Cκ, SEQ ID NO: 68) or λCL (Cλ, SEQ ID NO: 69), 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 Knob-Fc region has the amino acid sequence shown in SEQ ID NO: 66, the Hole-Fc region has the amino acid sequence shown in SEQ ID NO: 67, or any combination of (1) to (3).

[0006] Embodiment 3: A 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 the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or (2) SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or VL2 and VH2 have the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, or (2) SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

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

[0012] Embodiment 9: The bispecific antibodies according to Embodiment 7, wherein VL1, VH1, VL2, and VH2 each have the amino acid sequences shown in (1) SEQ ID NOs: 10, 11, 1, and 2, respectively; (2) SEQ ID NOs: 10, 11, 5, and 6, respectively; (3) SEQ ID NOs: 14, 15, 1, and 2, respectively; or (4) SEQ ID NOs: 14, 15, 5, and 6, respectively.

[0013] Embodiment 10: The bispecific antibodies 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 in (1) SEQ ID NOs: 18, 19, 3, and 20, respectively, (2) SEQ ID NOs: 21, 22, 3, and 20, respectively, (3) SEQ ID NOs: 18, 19, 7, and 23, respectively, or (4) SEQ ID NOs: 21, 22, 7, and 23, respectively.

[0014] Embodiment 11: (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 each specifically bind to human TNFα and human IL23p19, or each specifically binds to human IL23p19 and human TNFα, a bispecific antibody that specifically binds to human TNFα and human IL23p19.

[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 the human IgG1 CH region (SEQ ID NO: 58), or both (1) and (2).

[0016] Embodiment 13: A 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 each have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, or (2) SEQ ID NOs. 5 and 6, or VL2 and VH2 each have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, or (2) SEQ ID NOs. 14 and 15, or (3) SEQ ID NOs. 93 and 94, respectively.

[0018] Embodiment 15: The bispecific antibodies according to Embodiment 13, wherein VL1, VH1, VL2, and VH2 have the amino acid sequences shown in (1) SEQ ID NOs: 1, 2, 10, and 11, respectively; (2) SEQ ID NOs: 1, 2, 14, and 15, respectively; (3) SEQ ID NOs: 1, 2, 93, and 94, respectively; (4) SEQ ID NOs: 5, 6, 10, and 11, respectively; or (4) SEQ ID NOs: 5, 6, 14, and 15, respectively; and (4) SEQ ID NOs: 5, 6, 93, and 94, respectively.

[0019] Embodiment 16: The bispecific antibody according to Embodiment 13, wherein LC and HC each have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in (1) SEQ ID NOs: 3 and 30, respectively, (2) SEQ ID NOs: 3 and 31, respectively, (3) SEQ ID NOs: 7 and 32, respectively, (4) SEQ ID NOs: 7 and 33, respectively, or (5) SEQ ID NOs: 7 and 92, respectively.

[0020] Embodiment 17: A 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: A bispecific antibody according to Embodiment 17, wherein VL1 and VH1 have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, respectively, or (2) SEQ ID NOs. 14 and 15, respectively, or VL2 and VH2 have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, respectively, or (2) SEQ ID NOs. 5 and 6.

[0022] Embodiment 19: The bispecific antibodies according to Embodiment 17, wherein VL1, VH1, VL2, and VH2 each have the amino acid sequences shown in (1) SEQ ID NOs: 10, 11, 1, and 2, respectively; (2) SEQ ID NOs: 10, 11, 5, and 6, respectively; (3) SEQ ID NOs: 14, 15, 1, and 2, respectively; or (4) SEQ ID NOs: 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 the amino acid sequences shown in (1) SEQ ID NOs. 12 and 34, respectively, (2) SEQ ID NOs. 12 and 35, respectively, (3) SEQ ID NOs. 16 and 36, respectively, or (4) SEQ ID NOs. 16 and 37, respectively.

[0024] Embodiment 21: A bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) containing a first light chain variable domain (VL1), a second light chain variable domain (VL2), and a CL region, and (2) a heavy chain (HC) containing 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 each specifically bind to human TNFα and human IL23p19, or each specifically binds to human IL23p19 and human TNFα.

[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 the human IgG1 CH region (SEQ ID NO: 58), or both (1) and (2).

[0026] Embodiment 23: A 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: A bispecific antibody according to Embodiment 23, wherein VL1 and VH1 have (1) the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively, or (2) the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively, or VL2 and VH2 have (1) the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively, or (2) the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively.

[0028] Embodiment 25: The bispecific antibodies according to Embodiment 23, wherein VL1, VH1, VL2, and VH2 each have the amino acid sequences shown in (1) SEQ ID NOs: 1, 2, 10, and 11, respectively, (2) SEQ ID NOs: 1, 2, 14, and 15, respectively, (3) SEQ ID NOs: 5, 6, 10, and 11, respectively, or (4) SEQ ID NOs: 5, 6, 14, and 15, respectively.

[0029] Embodiment 26: The bispecific antibody according to Embodiment 23, wherein LC and HC each have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in (1) SEQ ID NOs. 46 and 47, respectively, (2) SEQ ID NOs. 48 and 49, respectively, (3) SEQ ID NOs. 50 and 51, respectively, or (4) SEQ ID NOs. 52 and 53, respectively.

[0030] Embodiment 27: A 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: A bispecific antibody according to Embodiment 27, wherein VL1 and VH1 have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, respectively, or (2) SEQ ID NOs. 14 and 15, respectively, or VL2 and VH2 have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, respectively, or (2) SEQ ID NOs. 5 and 6.

[0032] Embodiment 29: The bispecific antibodies according to Embodiment 27, wherein VL1, VH1, VL2, and VH2 each have the amino acid sequences shown in (1) SEQ ID NOs: 10, 11, 1, and 2, respectively; (2) SEQ ID NOs: 10, 11, 5, and 6, respectively; (3) SEQ ID NOs: 14, 15, 1, and 2, respectively; or (4) SEQ ID NOs: 14, 15, 5, and 6, respectively.

[0033] Embodiment 30: The bispecific antibody according to Embodiment 27, wherein LC and HC each have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in (1) SEQ ID NOs. 38 and 39, respectively, (2) SEQ ID NOs. 40 and 41, respectively, (3) SEQ ID NOs. 42 and 43, respectively, or (4) SEQ ID NOs. 44 and 45, respectively.

[0034] Embodiment 31: A bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) containing a light chain variable domain (VL) and a CL region, and (2) a heavy chain (HC) containing a heavy chain variable domain (VH) and a CH region, wherein (1) the VL / VH pair specifically binds to human IL23p19, and (2) HC further comprises a single heavy chain variable domain antibody (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 the human IgG1 CH region (SEQ ID NO: 58), or both (1) and (2).

[0036] Embodiment 33: A bispecific antibody according to Embodiment 31 or 32, wherein VL and VH have the amino acid sequences shown in (1) SEQ ID NOs: 10 and 11, respectively, or (2) SEQ ID NOs: 14 and 15, respectively.

[0037] Embodiment 34: VHH is a bispecific antibody according to any one of Embodiments 31 to 33, having the amino acid sequence shown in SEQ ID NO: 9.

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

[0039] Embodiment 36: The bispecific antibody according to Embodiment 35, wherein LC and HC each have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in (1) SEQ ID NOs. 12 and 54, respectively, or (2) SEQ ID NOs. 16 and 56, respectively.

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

[0041] Embodiment 38: The bispecific antibody according to Embodiment 37, wherein LC and HC each have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in (1) SEQ ID NOs. 12 and 55, respectively, or (2) SEQ ID NOs. 16 and 57, respectively.

[0042] Embodiment 39: A pharmaceutical composition comprising a 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, comprising administering a therapeutically effective dose of any one of Embodiments 1 to 38 of the bispecific antibody to the subject in need.

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

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

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

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

[0048] Embodiment 45: Use of a bispecific antibody as described in any one of Embodiments 1 to 38 as a pharmaceutical composition.

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

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

[0051] Embodiment 48: Use of a bispecific antibody according to any one of Embodiments 1 to 38 in the manufacture of a drug for treating an autoimmune disease.

[0052] Embodiment 49: Use of a bispecific antibody according to any one of Embodiments 1 to 38 in the manufacture of a drug for treating an inflammatory disease.

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

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

[0055] Embodiment 52: A vector comprising the polynucleotide described in Embodiment 50 or 51.

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

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

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

[0059] [Brief description of the drawing] [Figures 1A-1D] Figures of four different anti-TNFα / IL23P19 bispecific antibodies disclosed herein. (N) indicates the N-terminus and (C) indicates the C-terminus.

[0060] [Figure 1A] The "CrossMab-KIH" structure is composed of four distinct polypeptides: a first light chain (LC1), a first heavy chain (HC1), a second light chain (LC2), and a second heavy chain (HC2). Its composition is as follows: LC1:(N)-VL1-CH1-(C) HC1:(N)-VH1-CL region-Fc region (Knob)-(C) LC2:(N)-VL2-CL area-(C) HC2:(N)-VH2-CH1-Fc region (Hole)-(C).

[0061] [Figure 1B] This is the "IgG-ScFv" structure, which contains two pairs of identical light chains (LC) and heavy chains (HC), and its composition is as follows: LC:(N)-VL1-CL area-(C) HC:(N)-VH1-CH region-VH2-VL2-(C).

[0062] [Figure 1C] This is the "DVD-Ig" structure, which contains two pairs of identical light chains (LC) and heavy chains (HC), and its composition is as follows: LC:(N)-VL1-VL2-CL area-(C) HC:(N)-VH1-VH2-CH region-(C).

[0063] [Figure 1D] This is the "SMAB-VHH" structure, which contains two pairs of identical light chains (LC) and heavy chains (HC), and its composition is as follows: LC:(N)-VL1-CL area-(C) HC: (N)-VHH-VH-CH region-(C) or (N)-VH-CH region-VHH-(C).

[0064] [Figure 2] This chart summarizes the binding affinity of exemplary anti-TNFα / IL23p19 bispecific antibodies to TNFα and IL23p19, respectively.

[0065] [Figure 3] This chart illustrates how an exemplary anti-TNFα / IL23p19 bispecific antibody simultaneously binds to two antigens, namely human TNFα and IL23p19.

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

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

[0068] [Figure 6] This graph shows the effective inhibition of TNFα-induced U937 cell apoptosis by an exemplary anti-TNFα / IL23p19 bispecific antibody.

[0069] [Figure 7] This graph shows the effective inhibition of cytotoxicity in TNFα-inducible L929 cells by an exemplary anti-TNFα / IL23p19 bispecific antibody.

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

[0071] [Figure 9] This graph shows the effective inhibition of human TNFα-induced mRNA-6 production in mice by an exemplary anti-TNFα / IL23p19 bispecific antibody.

[0072] [Figure 10] A graph illustrating the effective inhibition of human IL-23-induced ear hyperplasia in mice by an exemplary anti-TNFα / IL23p19 bispecific antibody, where ear thickness, clinical PASI score, and ear histopathology score are evaluated.

[0073] [Overview of the prefecture] This disclosure provides novel bispecific antibodies that specifically bind to both human TNFα (TNFα) and the P19 subunit of human IL-23 (IL23p19). This disclosure also provides pharmaceutical compositions containing therapeutically effective amounts of these antibodies. Furthermore, this disclosure discloses the use of these antibodies and pharmaceutical compositions to alleviate autoimmunity and treat autoimmune and inflammatory diseases.

[0074] Before describing the present invention in further detail, it should be understood that the present invention is not limited to the specific embodiments described herein, and that the terms used herein are for describing specific embodiments and are not intended to limit them.

[0075] 1.Definition Unless otherwise specifically defined herein, the scientific and technical terms used in this invention should have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms include plural forms, and plural terms include singular forms. Generally, the terms and techniques used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry hybridization are well known and commonly used in the art.

[0076] The term "one" refers to one or more entities. For example, "one antibody" should be understood to mean one or more antibodies.

[0077] As used herein, the term "and / or" is deemed to be specifically disclosed regardless of whether each of the two designated features or components includes the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" means to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, as used in phrases such as "A, B and / or C," the term "and / or" means to include 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; A (alone); B (alone); and C (alone), respectively.

[0078] The terms “polypeptide,” “peptide,” “protein,” “polypeptide chain,” “peptide chain,” and their grammatical equivalents, as used interchangeably herein, refer to amino acid polymers of any length, which may be linear or branched. These may contain unnatural or modified amino acids, or may be interrupted by non-amino acids. Polypeptides, peptides, polypeptide chains, peptide chains, or proteins may also be modified, for example, by disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or any other operation or modification.

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

[0080] As used herein with respect to a protein or polypeptide having specific sequence features ("reference protein" or "reference polypeptide"), the term "mutant" refers to another protein or polypeptide having one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions compared to the reference protein or reference polypeptide. Amino acid sequence changes may be amino acid substitutions. Amino acid sequence changes may be conserved amino acid substitutions. Functional fragments or functional mutants of a protein or polypeptide maintain the basic structural and functional properties of the reference protein or polypeptide.

[0081] As used herein, “specific binding” means that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, faster, more persistently, with higher affinity, or in some combination of the above, compared to alternative substances including related and unrelated proteins. For example, a binding site (e.g., an antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified by immunoassay, ELISA, biolayer interferometry ("BLI"), SPR (e.g., Biacore) or other techniques well known to those skilled in the art. Generally, a specific response is at least twice the background signal or noise and may exceed 10 times the background. For a discussion of antibody specificity, see, for example, Paul, ed., 1989, FUNDAMENTAL IMMUNOLOGY SECOND EDITION, Raven Press, New York, pp. 332–336. A binding site that specifically binds to a target molecule may bind to the target molecule with higher affinity than its affinity to other molecules. In some embodiments, the binding site that specifically binds to the target molecule binds to the target molecule with an affinity at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times greater than its affinity to other molecules. In some embodiments, the binding site that specifically binds to a particular target molecule binds to a different molecule with an affinity so low that the binding cannot be detected using the tests described herein or other tests well known in the art. In some embodiments, "specific binding" is, for example, a binding site with a K of about 0.1 mM or less. D This means binding to a molecular target. In some embodiments, "specific binding" means that the polypeptide or molecule is about 10 M or less or about 1 μM or less. D This means binding to the target. In some embodiments, "specific binding" means that the polypeptide or molecule has a K content of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. DIt means binding to a target. Due to sequence identity between homologous proteins of different species, specific binding may involve a polypeptide or molecule that recognizes two or more proteins or targets. Similarly, due to homology within a specific region of the polypeptide sequences of different proteins, specific binding may involve a polypeptide or molecule that recognizes two or more proteins or targets. 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, i.e., does not necessarily need to be, exclusive binding, i.e., binding to a single target (it can include such binding). Thus, in some embodiments, a binding moiety (e.g., an antibody) can specifically bind to two or more targets. For example, an antibody may in certain cases contain two identical antigen-binding sites, each of which can specifically bind to the same epitope in two or more proteins. In certain alternative embodiments, the antibody may be bispecific and may contain at least two antigen-binding sites with different specificities.

[0082] As used herein, the term "binding affinity" generally refers to the strength of the sum of non-covalent binding 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 generally 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. Various methods for measuring binding affinity are well known in the art, and any of them can be used for the purposes of the present invention. Specific exemplary embodiments include the following. In some embodiments, the "K D " or "K D value" can be measured, for example, by assays well known in the art such as binding assays. K DThis can be measured by radioactive antigen binding validation (RIA) (Chen, et al., (1999) J. Mol Biol 293:865-881). D or K D The values ​​can also be measured using, for example, biolayer interferometry (BLI) with the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). Alternatively, K can be measured using, for example, surface plasmon resonance analysis (SPR) with Biacore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). D or K D The value can be measured.

[0083] In this specification, the terms “identical,” “percentage of identity,” and their grammatical equivalents, as used in the context of two or more polynucleotides or polypeptides, mean two or more sequences or subsequences having identical nucleotides or amino acid residues that are identical or have a specified percentage when comparing and aligning for maximum correspondence (with gaps introduced as necessary) and not considering any conserved amino acid substitutions as part of sequence identity. The identity ratio can be measured using sequence alignment software or algorithms or by visual inspection. Various algorithms and software used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their modifications. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning that when a sequence alignment algorithm is used to compare and align them for maximum correspondence, or when measured by visual inspection, 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. In some embodiments, the identity lies within a region of the amino acid sequence whose length is 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 in between. In some embodiments, the identity lies within a region longer than 60–80 residues, e.g., a region of at least about 80–100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared (e.g., the coding region of a target protein or antibody). In some embodiments, identity resides within a nucleotide sequence region having a length 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 of nucleotide sequence region between these.In some embodiments, the identity resides within a region longer than 60–80 bases, e.g., a region of at least about 80–1000 bases or more, and in some embodiments, the sequence is substantially identical over the entire length of the sequence being compared (e.g., the nucleotide sequence encoding the target protein).

[0084] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions are polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

[0085] Scope: Throughout this disclosure, various aspects of the present invention can be expressed in range form. It should be understood that descriptions in range form are merely for convenience and conciseness and should not be interpreted as arbitrarily limiting the scope of the present invention. Therefore, specific range descriptions should be considered to specifically disclose not only the individual numerical values ​​within that range, but also all possible subranges. For example, a range description such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numerical values ​​within that range such as 1, 2, 2.7, 3, 4, 5, 5.3, 6. This applies regardless of the width of the range.

[0086] In this specification, exemplary genes and polypeptides are described with reference to GenBank numbers, GI numbers, and / or sequence numbers. Those skilled in the art can easily 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 / ).

[0087] 2. Bispecific antibodies targeting TNFα and IL23p19 This specification provides bispecific antibodies that specifically bind to both human TNFα and human IL23p19. In some embodiments, the bispecific antibodies provided herein are monoclonal antibodies. In some embodiments, the bispecific antibodies provided herein are isolated. In some embodiments, the bispecific antibodies provided herein are substantially pure.

[0088] 2.1 Normal Case Tumor necrosis factor alpha (TNFα) is a pleomorphic homotrimeric cytokine. TNFα is primarily secreted by monocytes, macrophages, lymphocytes, endothelial cells, and fibroblasts. TNFα binds to two distinct receptors: TNFRI, expressed in almost all cell types, and TNFRII, expressed selectively in immune cells (CD4+ T cells, NK cells). TNFα is expressed in both soluble and transmembrane forms (the membrane-bound precursor form is hydrolyzed by the metalloproteinase TNFα-converting enzyme (TACE) to cleave into a soluble homotrimer). Both membrane-bound and soluble forms of the cytokine are biologically active. TNFα is a potent inflammatory response inducer. It promotes the production of inflammatory cytokines and chemokines, increases leukocyte recruitment and infiltration, and activates both innate and adaptive immunity. Therefore, TNFα is important in systemic inflammation, particularly in acute inflammatory responses. Excessive TNFα is 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). An exemplary amino acid sequence of human TNFα is as follows: MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQR EEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELR DNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRE TPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (Sequence ID 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 called IL23A. IL23 binds to a cell surface receptor consisting of the IL12 receptor β1 subunit and a unique IL23 receptor subunit. IL23R expression is limited to specific immune cell groups, primarily found in T cells (αβ and γδ TCR+) and subgroups of NK cells. IL23 is an upstream regulator of IL-6, IL-17, GM-CSF, and IL-22 in tissue inflammation. IL23 transmits signals downstream via TYK2 / JAK2-mediated STAT3 phosphorylation. IL23 promotes differentiation from untreated CD4+ T cells to pathogenic Th17 cells and stimulates iNKT cells, γδ T cells, and ILC3s to produce IL17 family cytokines. IL23 also promotes osteoclast production and bone resorption. IL23 promotes the activation of various inflammatory cells involved in the induction of chronic inflammation and regulates both the inflammatory response of memory / pathogenic T cells and the inflammatory activity of innate lymphoid cells. Therefore, increased IL23 production is considered 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. An exemplary amino acid sequence of the human IL23 p19 subunit is shown below; MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEG DEETTNDVPHIQCGGGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSP VGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVF AHGAATLSP (Sequence ID 75, Uniprot ID: Q9NPF7).

[0090] As used herein and as understood in the art, “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 located within the variable region of the immunoglobulin molecule. A “bispecific” antibody is an artificial hybrid antibody having two different antigen-binding sites that recognize and specifically bind to two different target antigens.

[0091] In this specification, the term “antibody” is used in its broadest sense and includes antibodies of different types and structures, including polyclonal antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecules containing antigen-binding sites (e.g., bivariate-region immunoglobulin molecules), as long as they exhibit the desired biological activity. Antibodies include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Based on the characteristics of the heavy chain constant domains, called α, δ, ε, γ, and μ respectively, antibodies may be any one of the five main immunoglobulins IgA, IgD, IgE, IgG, and IgM or their subtypes (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Unless otherwise specified, the term “antibody” as used herein includes the “antigen-binding fragment” of a complete antibody. The term “antigen-binding fragment” as used herein refers to a part or fragment of a complete antibody as the antigen-determining variable region of the complete 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 (HCAbs), light-chain antibodies (LCAbs), disulfide-bound scFv (dsscFv), double-chain antibodies, triple-chain antibodies, quadruple-chain antibodies, mini-antibodies, bivariable-domain antibodies (DVD), single-variable-domain antibodies (sdAbs, e.g., camel antibody, alpaca antibody), and bispecific or multispecific antibodies formed from a single-variable-domain (VHH) of a heavy-chain antibody and an antigen fragment.

[0092] The structure of immunoglobulins is well understood (see, for example, FUNDAMENTAL IMMUNOLOGY Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989))). Generally, immunoglobulins contain two pairs of polypeptide chains: one pair of light chains (L, low molecular weight) and one pair of heavy chains (H, high molecular weight), all four of which are interconnected by disulfide bonds.

[0093] Each light chain of an immunoglobulin typically contains a variable light chain region ("VL region") and a constant light chain region ("CL region"). Based on the amino acid sequence of the CL region, there are two distinct types of light chains: kappa (κ) and lambda (λ). The amino acid sequences of the CL region are well known in the art.

[0094] Each heavy chain typically contains a variable heavy chain region ("VH region") and a constant heavy chain region ("CH region"). The VH region may be one of five types, based on its amino acid sequence, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). When bound to a light chain, these different types of heavy chains produce five well-known antibody classes: IgA, IgD, IgE, IgG, and IgM. IgG has four subclasses: IgGl, IgG2, IgG3, and IgG4. The amino acid sequences of the CH regions of different antibody classes are well known in the art.

[0095] The CH region of immunoglobulins contains multiple domains. For example, the CH region of an IgG antibody consists of three domains: heavy chain constant domain 1 (CH1), heavy chain constant domain 2 (CH2), and heavy chain constant domain 3 (CH3). The highly flexible region between the CH1 and CH2 domains is called the "hinge region." The disulfide bond in the hinge region is part of the interaction between the two heavy chains in immunoglobulins. The "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain, which includes at least a portion of the constant region. In the isotypes of IgG, IgA, and IgD, the Fc region consists of the CH2 and CH3 domains, while the Fc region of IgM and IgE contains 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, as well as subtypes IgG1, IgG2, IgG3, and IgG4, are well known to those skilled in the art. While the boundaries of the Fc region of IgG heavy chains can 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 both the native sequence Fc region and the mutant Fc region. In some embodiments, the Fc domains of the two heavy chains of the bispecific antibodies provided herein may include paired modifications that promote binding to each other, instead of forming a homodimer.

[0097] Unless otherwise specified or inconsistent with the context, amino acid positions in the constant region follow EU numbering (Edelman et al., PNAS. 1969; 63:78-85, Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition. 1991 NIH Publication No. 91-3242). A list of exemplary amino acid sequences of the constant domain / region of human IgG antibodies is shown in Table 1 below.

[0098] [Table 1] TIFF2026514624000002.tif255167TIFF2026514624000003.tif255162TIFF2026514624000004.tif161169

[0099] The term "variable region" generally refers to a portion of the light or heavy chain of an immunoglobulin located at the amino terminus of the light or heavy chain, used for the binding and specificity of each particular antibody to a specific antigen. The variable region of the light chain is called the "light chain variable region" or "VL region" and contains at least one, generally one, "light chain variable domain" or "VL". The variable region of the heavy chain is called the "heavy chain variable region" or "VH region" and contains at least one, generally one, "heavy chain variable domain" or "VH". The sequence of variable domains differs significantly from antibody to antibody. VL and VH pairs can bind to form a binding site that specifically binds to the target antigen or epitope.

[0100] The VH and VL regions can be further subdivided into highly variable regions (or highly variable regions where the sequence and / or morphology of structurally defined loops may be highly variable), also known as complementarity-determining regions (CDRs), which are scattered throughout more conserved regions and are called framework regions (FRs). Sequence variability is concentrated in the CDRs, while less variable parts of the variable domain are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. Each VH and VL generally consists of three CDRs and four FRs arranged sequentially from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see Chothia and Lesk, J Mol Biol 1987;196:901-17).

[0101] A CDR represents one of three highly variable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VHβ-sheet framework, or one of three highly variable regions (L1, L2, or L3) within the non-framework region of an antibody VLβ-sheet framework. CDR regions are well known to those skilled in the art and are defined by various methods / systems. Such systems and / or definitions have been developed and improved over many years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines it as the most highly variable region 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 for antibody sequence analysis and CDR determination and are well known to those skilled in the art.

[0102] This specification discloses bispecific antibodies having different structures, including VH and VL that specifically bind to human TNFα or human IL23p19. While specific anti-human TNFα and anti-human IL23p19 VH / VL are described exemplary herein, those skilled in the art will understand that the bispecific antibodies disclosed herein are not limited to the exemplary VH / VL. This specification further expressly includes variants of the bispecific antibodies disclosed herein in which the exemplary anti-human TNFα and anti-human IL23p19 VH / VL are replaced with other anti-human TNFα and anti-human IL23p19 VH / VL. Each of the exemplary anti-human TNFα or anti-human IL23p19 VH / VL lists is shown in Table 2A or Table 2B.

[0103] [Table 2]

[0104] [Table 3]

[0105] 2.2CrossMab-KIH In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and human IL23p19 have a “CrossMab-KIH” structure, as shown in Figure 1A. The “knobs-into-holes” or “KIH” model promotes the formation of heterodimers of the designed bispecific antibodies rather than heavy-chain homodimers.

[0106] Modifications that promote Fc domain binding in bispecific antibodies include so-called "knob-into-hole" modifications, which involve a "knob" modification on one Fc domain and a "hole" modification on the other. Knob-into-hole techniques are described, for example, in US5,731,168, US7,695,936, Ridgway et al., Prot. Eng. 9, 617-621 (1996) and Carter, J Immunol. Meth. 248, 7-15 (2001). Generally, these methods promote heterodimer formation and hinder homodimer formation by introducing a projection ("knob") at the interface of the first Fc ("knob-Fc") and a corresponding cavity ("hole") at the interface of the second Fc ("hole-Fc"), so that the projection is located within the cavity. The protrusions are constructed by replacing the smaller amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protrusions are generated at the interface of the second polypeptide by replacing the larger amino acid side chains with smaller amino acids (e.g., alanine or threonine).

[0107] Therefore, the "Knob-Fc region" and the "Hole-Fc region" are designed to form a heterodimer pair. In the Knob-Fc region, amino acids in the CH3 domain are substituted with amino acid residues having a larger side chain volume to generate a protrusion that may be located within the CH3 domain in the cavity of the CH3 domain of the Hole-Fc region, and in the Hole-Fc region, amino acid residues in the CH3 domain are substituted with amino acid residues having a smaller side chain volume to generate a cavity within the CH3 domain in which a protrusion within the CH3 domain of the first subunit can be located. 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 protrusions and cavities can be produced by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0108] In some embodiments, the 366th threonine residue in the Knob-Fc region is substituted with a tryptophan residue (T366W), the 407th tyrosine residue in the Hole-Fc region is substituted with a valine residue (Y407V), and optionally, the 366th threonine residue is substituted with a serine residue (T366S), and the 368th leucine residue is substituted with an alanine residue (L368A). In some embodiments, the 354th serine residue in the Knob-Fc region is substituted with a cysteine ​​residue (S354C), or the 356th glutamic acid residue is substituted with a cysteine ​​residue (E356C), and the 349th tyrosine residue in the Hole-Fc region (Y349C) is substituted with a cysteine ​​residue. In some embodiments, the Knob-Fc region includes amino acid substitutions S354C and T366W, and the Hole-Fc region includes amino acid substitutions Y349C, T366S, L368A and Y407V. All amino acid residues are numbered according to the EU index.

[0109] By swapping one side of CL and CH1, the CrossMab design is used to resolve the BsAb light chain mismatch. By swapping one side of the heavy chain and light chain domain, the BsAb light chain can be assembled correctly.

[0110] Therefore, in some embodiments, the bispecific antibodies provided herein may 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 antibodies can employ a KIH design, where HC1 includes a knob-Fc region and HC2 includes a hole-Fc region. As another option, in some embodiments, HC2 may include a knob-Fc region and HC1 may include a hole-Fc region. Furthermore, light chain mismatch is avoided by exchanging the light chain constant region (CL region) of LC1 with the heavy chain constant domain 1 (CH1) of HC1. Accordingly, the bispecific antibodies provided herein may have LC1, HC1, LC2 and HC2, where (1) the LC1 / HC1 pair has an exchanged constant domain, (2) the LC2 / HC2 pair has a normal constant domain, and (3) the HC1 / HC2 has a KIH structure. In some embodiments, HC1 has a knob-Fc region and HC2 has a hole-Fc region. In some embodiments, HC1 has a hole-Fc region and HC2 has a knob-Fc region.

[0111] In some embodiments, the bispecific antibodies provided herein that specifically bind to NFα and human IL23p19 include: (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 variable domain (VH2), a second CH1 domain, and a Hole-F The polypeptide has four polypeptides, each containing a c-region and a double chain (HC2), 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 human Fc region variant with a T366W substitution (numbered by the EU index), and the Hole-Fc region is a human Fc region variant with a Y407V substitution (numbered by the EU index).

[0112] The amino acid sequences of the CH1, CL, and Fc regions (CH2 and CH3) of the bispecific antibodies disclosed herein may be derived from any suitable source, such as the constant regions of antibodies like IgG1, IgG2, IgG3, or IgG4. The amino acid sequences of the constant regions of the heavy and light chains of antibodies are well known in the art, for example, 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 domain and constant region of the bispecific antibodies provided herein are derived from human IgG. In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG1. In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG2. In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG3. In some embodiments, the constant domain and constant region of the bispecific antibodies provided herein are derived from human IgG4. In some embodiments, the amino acid sequences of the CH1, CL region and Fc region (hinge, CH2 and CH3) of the bispecific antibodies disclosed herein may include one or more amino acid substitutions that differ from wild-type immunoglobulin, for example, one or more amino acid substitutions in wild-type IgG1 or IgG4. Such substitutions are well known in the art (see, for example, US7704497, US7083784, US6821505, US8323962, US6737056 and US7416727).

[0114] In some embodiments, the first and second CH1 domains may 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 may be κCL(Cκ, SEQ ID NO: 68). In some embodiments, the CL region may 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 region 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 may 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 may be a variant of the Fc region of human IgG1. In some embodiments, the Knob-Fc region is human IgG1 Fc having the T366W substitution. In some embodiments, the Hole-Fc region is human IgG1 Fc having the Y407T substitution. In some embodiments, the Knob-Fc and Hole-Fc regions may further include the S354C and Y349C substitutions, respectively. In some embodiments, the Knob-Fc and Hole-Fc regions may further include the E356C and Y349C substitutions, respectively. In some embodiments, the Hole-Fc region may further include the T366S and L368A substitutions. All amino acid residues are numbered according to the EU index. In some embodiments, the Knob-Fc region may have the amino acid sequence shown in SEQ ID NO: 66. In some embodiments, the Hole-Fc region may have the amino acid sequence shown in SEQ ID NO: 67. A list of exemplary Fc sequences in the KIH model is shown in Table 3 below.

[0118] [Table 4]

[0119] In some embodiments of the bispecific antibodies provided herein, (1) the first CL region is κCL (Cκ, SEQ ID NO: 68) or λCL (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 and second CH1 domains are human IgG1 CH1 domains (SEQ ID NO: 61); or (3) the Knob-Fc region has the amino acid sequence shown in SEQ ID NO: 66, and the Hole-Fc region has the amino acid sequence shown in SEQ ID NO: 67; or any combination of (1) to (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 may be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL1 and VH1 are VL and VH of adalimumab, having the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL1 and VH1 are VL and VH of golimumab, having the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively. The VL2 / VH2 pair may be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL2 and VH2 are VL and VH of guselkumab, having the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL2 and VH2 are VL and VH of tildrakizumab, having the amino acid sequences shown in SEQ ID NOs: 14 and 15, respectively.

[0121] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, respectively, or (2) SEQ ID NOs. 5 and 6, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, respectively, or (2) SEQ ID NOs. 14 and 15, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively.

[0122] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in (1) SEQ ID NOs: 1, 2, 10, and 11, respectively; (2) SEQ ID NOs: 1, 2, 14, and 15, respectively; (3) SEQ ID NOs: 5, 6, 10, and 11, respectively; or (4) SEQ ID NOs: 5, 6, 14, and 15, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 1, 2, 10, and 11, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 1, 2, 14, and 15, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 5, 6, 10, and 11, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 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 may be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL1 and VH1 are VL and VH of guselkumab, having the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL1 and VH1 are VL and VH of tildrakizumab, having the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively. The VL2 / VH2 pair may be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL2 and VH2 are VL and VH of adalimumab, having the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL2 and VH2 are VL and VH of golimumab, having the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively.

[0124] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, respectively, or (2) SEQ ID NOs. 14 and 15, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, respectively, or (2) SEQ ID NOs. 5 and 6, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively.

[0125] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in (1) SEQ ID NOs: 10, 11, 1, and 2, respectively; (2) SEQ ID NOs: 10, 11, 5, and 6, respectively; (3) SEQ ID NOs: 14, 15, 1, and 2, respectively; or (4) SEQ ID NOs: 14, 15, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 10, 11, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 10, 11, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 14, 15, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 14, 15, 5, and 6, respectively. A list of examples of bispecific antibodies, VL1, VH1, VL2, and VH2, is shown in Table 4A below.

[0126] [Table 5] TIFF2026514624000009.tif255162TIFF2026514624000010.tif255162TIFF2026514624 000011.tif255162TIFF2026514624000012.tif255167TIFF2026514624000013.tif25516 2TIFF2026514624000014.tif255162TIFF2026514624000015.tif255162TIFF2026514624 000016.tif255167TIFF2026514624000017.tif255162TIFF2026514624000018.tif69169

[0127] In some embodiments, the Specified provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, wherein LC1, HC1, LC2, and HC2 each have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in (1) SEQ ID NOs: 18, 19, 3, and 20, respectively; (2) SEQ ID NOs: 21, 22, 7, and 23, respectively; (3) SEQ ID NOs: 21, 22, 7, and 23, respectively; (5) SEQ ID NOs: 24, 25, 12, and 26, respectively; (6) SEQ ID NOs: 27, 28, 12, and 26, respectively; (7) SEQ ID NOs: 24, 25, 16, and 29, respectively; or (8) SEQ ID NOs: 27, 28, 16, and 29, respectively.

[0128] In some embodiments, this specification provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 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 NOs. In some embodiments, LC1 has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC1 has the amino acid sequence shown in SEQ ID NOs. 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 in 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 in SEQ ID NO: 20. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 18, 19, 3, and 20, respectively.In some embodiments, this specification provides a bispecific antibody represented by A1, where LC1, HC1, LC2, and HC2 have the amino acid sequences shown in SEQ ID NOs. 18, 19, 3, and 20, respectively.

[0129] In some embodiments, this specification provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 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 with SEQ ID NOs. In some embodiments, LC1 has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC1 has the amino acid sequence shown in SEQ ID NOs. 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: 3. In some embodiments, LC2 has the amino acid sequence shown in 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 in SEQ ID NO: 20. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 21, 22, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 21, 22, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 21, 22, 3, and 20, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 21, 22, 3, and 20, respectively.In some embodiments, this specification provides a bispecific antibody represented by A2, where LC1, HC1, LC2, and HC2 have the amino acid sequences shown in SEQ ID NOs: 21, 22, 3, and 20, respectively.

[0130] In some embodiments, this specification provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 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 NOs. In some embodiments, LC1 has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC1 has the amino acid sequence shown in SEQ ID NOs. 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 in 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 in SEQ ID NO: 23. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 18, 19, 7, and 23, respectively.In some embodiments, this specification provides a bispecific antibody represented by A3, where LC1, HC1, LC2, and HC2 have the amino acid sequences shown in SEQ ID NOs: 18, 19, 7, and 23, respectively.

[0131] In some embodiments, this specification provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 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 NOs. In some embodiments, LC1 has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC1 has the amino acid sequence shown in SEQ ID NOs. 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 in 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 in SEQ ID NO: 23. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 21, 22, 7, and 23, respectively.In some embodiments, this specification provides a bispecific antibody represented by A4, where LC1, HC1, LC2, and HC2 have the amino acid sequences shown in SEQ ID NOs: 21, 22, 7, and 23, respectively.

[0132] In some embodiments, this specification provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 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 NOs. In some embodiments, LC1 has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC1 has the amino acid sequence shown in SEQ ID NOs. 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 in 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 in SEQ ID NO: 26. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 24, 25, 12, and 26, respectively.In some embodiments, this specification provides a bispecific antibody represented by A5, where LC1, HC1, LC2, and HC2 have the amino acid sequences shown in SEQ ID NOs: 24, 25, 12, and 26, respectively.

[0133] In some embodiments, this specification provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 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 with SEQ ID NOs. In some embodiments, LC1 has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC1 has the amino acid sequence shown in SEQ ID NOs. 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: 12. In some embodiments, LC2 has the amino acid sequence shown in 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 in SEQ ID NO: 26. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 27, 28, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 27, 28, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 27, 28, 12, and 26, respectively. In some embodiments, LC1, HC1, LC2, and HC2 have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 27, 28, 12, and 26, respectively.In some embodiments, this specification provides a bispecific antibody represented by A6, where LC1, HC1, LC2, and HC2 have the amino acid sequences shown in SEQ ID NOs. 27, 28, 12, and 26, respectively.

[0134] In some embodiments, this specification provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 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 NOs. In some embodiments, LC1 has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC1 has the amino acid sequence shown in SEQ ID NOs. 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 in 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 in SEQ ID NO: 29. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs: 24, 25, 16, and 29, respectively.In some embodiments, this specification provides a bispecific antibody represented by A7, where LC1, HC1, LC2, and HC2 have the amino acid sequences shown in SEQ ID NOs: 24, 25, 16, and 29, respectively.

[0135] In some embodiments, this specification provides bispecific antibodies having LC1, HC1, LC2, and HC2 that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 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 NOs. In some embodiments, LC1 has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC1 has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC1 has the amino acid sequence shown in SEQ ID NOs. 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 in 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 in SEQ ID NO: 29. In some embodiments, LC1, HC1, LC2, and HC2 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 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 in SEQ ID NOs: 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 in SEQ ID NOs. 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 in SEQ ID NOs. 27, 28, 16, and 29, respectively.In some embodiments, this specification provides a bispecific antibody represented by A8, where LC1, HC1, LC2, and HC2 have the amino acid sequences shown in SEQ ID NOs. 27, 28, 16, and 29, respectively.

[0136] 2.3IgG-ScFv In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and human IL23p19 have an "IgG-ScFv" structure as shown in Figure 1B. As shown in the figure, the scFv, which includes a VH / VL pair that specifically binds to the first antigen, is linked to the CH3 domain of IgG that specifically binds to the second antigen. In some embodiments, the N-terminus of the scFv is linked to the C-terminus of the IgG heavy chain. In some embodiments, the N-terminus of the scFv is linked to the C-terminus of the CH3 domain of IgG. In some embodiments, the N-terminus of the scFv is linked to the C-terminus of the IgG light chain. In some embodiments, the N-terminus of the scFv is linked to the C-terminus of the VL region of IgG. In some embodiments, the scFv has a linker linking VH and VL. In some embodiments of the scFv, the N-terminus of VH is linked to the C-terminus of VL. In some embodiments of the scFv, the N-terminus of VL is linked to the C-terminus of VH.

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

[0138] Accordingly, this specification provides a bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) including a first light chain variable domain (VL1) and a CL region, and (2) a heavy chain (HC) including 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 binds to human IL23p19 and human TNFα, respectively. In some embodiments, the bispecific antibody provided herein has two identical LC and HC pairs.

[0139] As another option, this specification provides a bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) containing 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) containing 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 antibody provided herein has two identical LC and HC pairs.

[0140] As another option, this specification provides a bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) containing a first light chain variable domain (VL1) and a CL region, and (2) a heavy chain (HC) containing 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), where the VL1 / VH1 pair and the VL2 / VH2 pair specifically bind to human TNFα and human IL23p19, respectively, or specifically binds to human IL23p19 and human TNFα, respectively. In some embodiments, the bispecific antibody provided herein has two identical LC and HC pairs.

[0141] As another option, this specification provides a bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) containing 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) containing 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 antibody provided herein has two identical LC and HC pairs.

[0142] The amino acid sequences of the CL and CH regions of the bispecific antibodies disclosed herein may originate from any suitable source and may be, for example, the constant regions of an antibody 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 region and Fc region (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein may include one or more amino acid substitutions different from those in wild-type immunoglobulin, for example, one or more amino acid substitutions in wild-type IgG1 or IgG4. Such substitutions are well known in the art (see, for example, US7704497, US7083784, US6821505, US8323962, US6737056, and US7416727).

[0143] In some embodiments, the CH region may be independently 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 may be the human IgG1 CH region (SEQ ID NO: 58).

[0144] In some embodiments, the CL region may be κCL(Cκ, SEQ ID NO: 68). In some embodiments, the CL region may 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 a CH region or a 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, the VL2 and VH2 of 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 may be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL1 and VH1 are VL and VH of adalimumab, having the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL1 and VH1 are VL and VH of golimumab, having the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively. The VL2 / VH2 pair may be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL2 and VH2 are VL and VH of guselkumab, having the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL2 and VH2 are VL and VH of guselkumab, having the amino acid sequences shown in SEQ ID NOs. 93 and 94, respectively. In some embodiments, VL2 and VH2 are VL and VH of tildrakizumab, having the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively.

[0149] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, respectively, or (2) SEQ ID NOs. 5 and 6, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, respectively, (2) SEQ ID NOs. 14 and 15, respectively, or (3) SEQ ID NOs. 93 and 94, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 93 and 94, respectively.

[0150] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in (1) SEQ ID NOs: 1, 2, 10, and 11, respectively; (2) SEQ ID NOs: 1, 2, 14, and 15, respectively; (3) SEQ ID NOs: 1, 3, 93, and 94, respectively; (4) SEQ ID NOs: 5, 6, 10, and 11, respectively; (5) SEQ ID NOs: 5, 6, 14, and 15, respectively; or (6) SEQ ID NOs: 5, 6, 93, and 94, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 1, 2, 10, and 11, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 1, 2, 14, and 15, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 1, 2, 93, and 94, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs. 5, 6, 10, and 11, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs. 5, 6, 14, and 15, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs. 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 may be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL1 and VH1 are VL and VH of guselkumab, having the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL1 and VH1 are VL and VH of tildrakizumab, having the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively. The VL2 / VH2 pair may be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL2 and VH2 are VL and VH of adalimumab, having the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL2 and VH2 are VL and VH of golimumab, having the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively.

[0152] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, respectively, or (2) SEQ ID NOs. 14 and 15, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, respectively, or (2) SEQ ID NOs. 5 and 6, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively.

[0153] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in (1) SEQ ID NOs: 10, 11, 1, and 2, respectively; (2) SEQ ID NOs: 10, 11, 5, and 6, respectively; (3) SEQ ID NOs: 14, 15, 1, and 2, respectively; or (4) SEQ ID NOs: 14, 15, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 10, 11, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 10, 11, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 14, 15, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 14, 15, 5, and 6, respectively. A list of exemplary LCs and HCs of bispecific antibodies is shown in Table 4B below.

[0154] [Table 6] TIFF2026514624000020.tif255162TIFF2026514624000021.tif255162TIFF2026514624000022.tif255162TIFF20265146240 00023.tif255162TIFF2026514624000024.tif255162TIFF2026514624000025.tif255162TIFF2026514624000026.tif197169

[0155] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where LC and HC each have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in (1) SEQ ID NOs: 3 and 30, respectively, (2) SEQ ID NOs: 3 and 31, respectively, (3) SEQ ID NOs: 7 and 32, respectively, (4) SEQ ID NOs: 7 and 33, respectively, (5) SEQ ID NOs: 12 and 34, respectively, (6) SEQ ID NOs: 12 and 35, respectively, (7) SEQ ID NOs: 16 and 36, respectively, (8) SEQ ID NOs: 16 and 37, respectively, or (8) SEQ ID NOs: 7 and 92, respectively.

[0156] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 3 and 30, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 3. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 3. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 3. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 3. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 3 and 30, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 3 and 30, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 3 and 30, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 3 and 30, respectively. In some embodiments, this specification provides a bispecific antibody represented by B1, where LC and HC have the amino acid sequences shown in SEQ ID NOs: 3 and 30, respectively.

[0157] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs: 3 and 31, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs: 3. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs: 3. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs: 31. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs: 31. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 3 and 31, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 3 and 31, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 3 and 31, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 3 and 31, respectively. In some embodiments, this specification provides a bispecific antibody represented by B2, where LC and HC have the amino acid sequences shown in SEQ ID NOs: 3 and 31, respectively.

[0158] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NO: 7 and 32, respectively. In some embodiments, 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, LC has the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, 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, HC has the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 7 and 32, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NO: 7 and 32, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 7 and 32, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 7 and 32, respectively. In some embodiments, this specification provides a bispecific antibody represented by B3, where LC and HC have the amino acid sequences shown in SEQ ID NOs: 7 and 32, respectively.

[0159] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NO: 7 and 92, respectively. In some embodiments, 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, LC has the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 92. In some embodiments, HC has the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 7 and 92, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NO: 7 and 92, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 7 and 92, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 7 and 92, respectively. In some embodiments, this specification provides a bispecific antibody represented by B3-1, where LC and HC have the amino acid sequences shown in SEQ ID NOs: 7 and 92, respectively.

[0160] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NO: 7 and 33, respectively. In some embodiments, 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, LC has the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, 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, HC has the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 7 and 33, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NO: 7 and 33, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 7 and 33, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 7 and 33, respectively. In some embodiments, this specification provides a bispecific antibody represented by B3-1, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 7 and 33, respectively.

[0161] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 12 and 34, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 12. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 12. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 34. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 34. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 34, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 34, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 34, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 34, respectively. In some embodiments, this specification provides a bispecific antibody represented by B5, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 12 and 34, respectively.

[0162] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 12 and 35, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 35, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 35, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 35, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 35, respectively. In some embodiments, this specification provides a bispecific antibody represented by B6, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 12 and 35, respectively.

[0163] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 16 and 36, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 16 and 36, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 36, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 36, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 36, respectively. In some embodiments, this specification provides a bispecific antibody represented by B7, where LC and HC have the amino acid sequences shown in SEQ ID NOs: 16 and 36, respectively.

[0164] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 16 and 37, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 16. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 16. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 37. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 37. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 16 and 37, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 37, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 37, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 37, respectively. In some embodiments, this specification provides a bispecific antibody represented by B8, where LC and HC have the amino acid sequences shown in SEQ ID NOs: 16 and 37, respectively.

[0165] 2.4 DVD-IG In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and human IL23p19 have a “dual variable domain-immunoglobulin” or “DVD-Ig” structure, as shown in Figure 1C. DVD-Ig is a symmetrical structure with four antigen-binding sites that can simultaneously bind to two different targets. The DVD-Ig structure includes an Fc region, and each antibody arm links two variable regions using a flexible short peptide.

[0166] Accordingly, this specification provides a bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) containing a first light chain variable domain (VL1), a second light chain variable domain (VL2), and a CL region, and (2) a heavy chain (HC) containing 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 binds to human IL23p19 and human TNFα, respectively.

[0167] The amino acid sequences of the CL and CH regions of the bispecific antibodies disclosed herein may be derived from any suitable source, for example, 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 region, and Fc region (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein may include one or more amino acid substitutions different from those in wild-type immunoglobulin, for example, one or more amino acid substitutions in wild-type IgG1 or IgG4. Such substitutions are well known in the art (see, for example, US7704497, US7083784, US6821505, US8323962, US6737056, and US7416727).

[0168] In some embodiments, the CH region may be independently 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 may be the human IgG1 CH region (SEQ ID NO: 58).

[0169] In some embodiments, the CL region may be κCL(Cκ, SEQ ID NO: 68). In some embodiments, the CL region may 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 the 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 may be linked to each other via linkers. 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 may be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL1 and VH1 are VL and VH of adalimumab, having the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL1 and VH1 are VL and VH of golimumab, having the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively. The VL2 / VH2 pair may be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL2 and VH2 are VL and VH of guselkumab, having the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL2 and VH2 are VL and VH of tildrakizumab, having the amino acid sequences shown in SEQ ID NOs: 14 and 15, respectively.

[0173] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, respectively, or (2) SEQ ID NOs. 5 and 6, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, respectively, or (2) SEQ ID NOs. 14 and 15, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively.

[0174] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in (1) SEQ ID NOs: 1, 2, 10, and 11, respectively; (2) SEQ ID NOs: 1, 2, 14, and 15, respectively; (3) SEQ ID NOs: 5, 6, 10, and 11, respectively; or (4) SEQ ID NOs: 5, 6, 14, and 15, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 1, 2, 10, and 11, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 1, 2, 14, and 15, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 5, 6, 10, and 11, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 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 may be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL1 and VH1 are VL and VH of guselkumab, having the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL1 and VH1 are VL and VH of tildrakizumab, having the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively. The VL2 / VH2 pair may be any VL / VH pair that specifically binds to human TNFα. In some embodiments, VL2 and VH2 are VL and VH of adalimumab, having the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL2 and VH2 are VL and VH of golimumab, having the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively.

[0176] In some embodiments of the bispecific antibodies provided herein, VL1 and VH1 have the amino acid sequences shown in (1) SEQ ID NOs. 10 and 11, respectively, or (2) SEQ ID NOs. 14 and 15, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 10 and 11, respectively. In some embodiments, VL1 and VH1 have the amino acid sequences shown in SEQ ID NOs. 14 and 15, respectively. In some embodiments of the bispecific antibodies provided herein, VL2 and VH2 have the amino acid sequences shown in (1) SEQ ID NOs. 1 and 2, respectively, or (2) SEQ ID NOs. 5 and 6, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 1 and 2, respectively. In some embodiments, VL2 and VH2 have the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively.

[0177] In some embodiments of the bispecific antibodies provided herein, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in (1) SEQ ID NOs: 10, 11, 1, and 2, respectively; (2) SEQ ID NOs: 10, 11, 5, and 6, respectively; (3) SEQ ID NOs: 14, 15, 1, and 2, respectively; or (4) SEQ ID NOs: 14, 15, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 10, 11, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 10, 11, 5, and 6, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 14, 15, 1, and 2, respectively. In some embodiments, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in SEQ ID NOs: 14, 15, 5, and 6, respectively. A list of exemplary LCs and HCs of bispecific antibodies is shown in Table 4C below.

[0178] [Table 7] TIFF2026514624000028.tif255162TIFF2026514624000029.tif255162TIFF2026514624000030.tif255162TIFF2026514624 000031.tif255162TIFF2026514624000032.tif255162TIFF2026514624000033.tif255162TIFF2026514624000034.tif99169

[0179] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where LC and HC each have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences shown in (1) SEQ ID NOs. 38 and 39, respectively, (2) SEQ ID NOs. 40 and 41, respectively, (3) SEQ ID NOs. 42 and 43, respectively, (4) SEQ ID NOs. 44 and 45, respectively, (5) SEQ ID NOs. 46 and 47, respectively, (6) SEQ ID NOs. 48 and 49, respectively, (7) SEQ ID NOs. 50 and 51, respectively, or (8) SEQ ID NOs. 52 and 53, respectively.

[0180] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 38 and 39, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 38. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 38. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 39. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 39. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 38 and 39, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 38 and 39, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 38 and 39, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 38 and 39, respectively. In some embodiments, this specification provides a bispecific antibody represented by C1, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 38 and 39, respectively.

[0181] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 40 and 41, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 40. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 41. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 41. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 40 and 41, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 40 and 41, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 40 and 41, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 40 and 41, respectively. In some embodiments, this specification provides a bispecific antibody represented by C2, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 40 and 41, respectively.

[0182] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 42 and 43, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 42. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 43. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 43. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 42 and 43, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 42 and 43, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 42 and 43, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 42 and 43, respectively. In some embodiments, this specification provides a bispecific antibody represented by C3, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 42 and 43, respectively.

[0183] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 44 and 45, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 44 and 45, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 44 and 45, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 44 and 45, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 44 and 45, respectively. In some embodiments, this specification provides a bispecific antibody represented by C4, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 44 and 45, respectively.

[0184] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 46 and 47, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 46. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 47. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 47. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 46 and 47, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 46 and 47, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 46 and 47, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 46 and 47, respectively. In some embodiments, this specification provides a bispecific antibody represented by C5, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 46 and 47, respectively.

[0185] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 48 and 49, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 48. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 48. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 49. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 49. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 48 and 49, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 48 and 49, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 48 and 49, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 48 and 49, respectively. In some embodiments, this specification provides a bispecific antibody represented by C6, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 48 and 49, respectively.

[0186] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 50 and 51, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. 50. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 51. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. 51. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 50 and 51, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 50 and 51, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 50 and 51, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 50 and 51, respectively. In some embodiments, this specification provides a bispecific antibody represented by C7, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 50 and 51, respectively.

[0187] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 52 and 53, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 52 and 53, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 52 and 53, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 52 and 53, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 52 and 53, respectively. In some embodiments, this specification provides a bispecific antibody represented by C8, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 52 and 53, respectively.

[0188] 2.5SMAB In some embodiments, the bispecific antibodies provided herein that specifically bind to human TNFα and human IL23p19 have a “monoclonal antibody fused to a single-domain antibody” or “SMAB” structure as shown in Figure 1D. The single-domain antibody may be a single heavy-chain variable-domain antibody ("VHH"). The VHH may be ligated to the heavy or light chain of IgG. In some embodiments, the VHH is ligated to the heavy chain of IgG. In some embodiments, the VHH is ligated to the N-terminus of the IgG heavy chain. In some embodiments, the VHH is ligated to the C-terminus of the IgG heavy chain. In some embodiments, the VHH is ligated to the IgG light chain. In some embodiments, the VHH is ligated to the N-terminus of the IgG light chain. In some embodiments, the VHH is ligated to the C-terminus of the IgG heavy chain.

[0189] Accordingly, this specification provides a bispecific antibody that specifically binds 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 VHH that specifically binds to human TNFα. In some embodiments, VHH is ligated to the N-terminus of the HC. In some embodiments, VHH is ligated to the C-terminus of the HC.

[0190] This specification provides a bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) containing a light chain variable domain (VL) and a CL region, and (2) a heavy chain (HC) containing 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 VHH, which specifically binds to human TNFα. In some embodiments, VHH is ligated to the N-terminus of the HC. In some embodiments, VHH is ligated to the C-terminus of the HC.

[0191] This specification provides a bispecific antibody that specifically binds 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 VHH, which specifically binds to human TNFα. In some embodiments, VHH is ligated to the N-terminus of the HC. In some embodiments, VHH is ligated to the C-terminus of the LC.

[0192] This specification provides a bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) containing a light chain variable domain (VL) and a CL region, and (2) a heavy chain (HC) containing 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 includes VHH that specifically binds to human IL23p19. In some embodiments, VHH is ligated to the N-terminus of the LC. In some embodiments, VHH is ligated to the C-terminus of the LC.

[0193] The amino acid sequences of the CL and CH regions of the bispecific antibodies disclosed herein may be derived from any suitable source, for example, 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 region, and Fc region (hinge, CH2, and CH3) of the bispecific antibodies disclosed herein may include one or more amino acid substitutions different from those in wild-type immunoglobulin, for example, one or more amino acid substitutions in wild-type IgG1 or IgG4. Such substitutions are well known in the art (see, for example, US7704497, US7083784, US6821505, US8323962, US6737056, and US7416727).

[0194] In some embodiments, the CH region may be independently 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 may be the human IgG1 CH region (SEQ ID NO: 58).

[0195] In some embodiments, the CL region may be κCL(Cκ, SEQ ID NO: 68). In some embodiments, the CL region may 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, VHH is linked to the IgG heavy chain via a linker. In some embodiments, the C-terminus of VHH is linked to the N-terminus of VH via a linker. In some embodiments, the N-terminus of VHH is linked to the C-terminus of VH 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 VHH is linked to the N-terminus of VL via a linker. In some embodiments, the N-terminus of VHH is linked to the C-terminus of CL 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, this specification provides a bispecific antibody that specifically binds to human TNFα and human IL23p19, comprising (1) a light chain (LC) including a light chain variable domain (VL) and a CL region, and (2) a heavy chain (HC) including 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 includes VHH that specifically binds to human TNFα. In some embodiments, VHH is ligated to the N-terminus of HC. In some embodiments, VHH is ligated to the C-terminus of HC. The VL / VH pair may be any VL / VH pair that specifically binds to human IL23p19. In some embodiments, VL and VH are VL and VH of guselkumab, having the amino acid sequences shown in SEQ ID NOs: 10 and 11, respectively. In some embodiments, VL and VH are VL and VH of tildrakizumab, having the amino acid sequences shown in SEQ ID NOs: 14 and 15, respectively. VHH may be any VHH that specifically binds to human TNFα. In some embodiments, VHH may be ozoralizumab (SEQ ID NO: 9).

[0199] In some embodiments of the bispecific antibodies provided herein, VL and VH have the amino acid sequences shown in SEQ ID NOs: 10 and 11, respectively, and VHH has the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs: 14 and 15, respectively, and VHH has the amino acid sequence shown in SEQ ID NO: 9. A list of exemplary LC and HC of the bispecific antibodies is shown in Table 4D below.

[0200] [Table 8] TIFF2026514624000036.tif255162TIFF2026514624000037.tif255167TIFF2026514624000038.tif24169

[0201] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 12 and 54, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 54, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 54, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 54, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 54, respectively. In some embodiments, this specification provides a bispecific antibody represented by D1, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 12 and 54, respectively.

[0202] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 12 and 55, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 55, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 55, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 55, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 12 and 55, respectively. In some embodiments, this specification provides a bispecific antibody represented as D2, where LC and HC have the amino acid sequences shown in SEQ ID NOs. 12 and 55, respectively.

[0203] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 16 and 56, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 16 and 56, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 56, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 56, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 56, respectively. In some embodiments, this specification provides a bispecific antibody represented by D3, where LC and HC have the amino acid sequences shown in SEQ ID NOs: 16 and 56, respectively.

[0204] In some embodiments, this specification provides bispecific antibodies having LC and HC that specifically bind to human TNFα and human IL23p19, where 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 in SEQ ID NOs. 16 and 57, respectively. In some embodiments, LC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, LC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, HC has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. In some embodiments, HC has the amino acid sequence shown in SEQ ID NOs. In some embodiments, LC and HC have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 16 and 57, respectively. In some embodiments, LC and HC have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 57, respectively. In some embodiments, LC and HC have at least 98% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 57, respectively. In some embodiments, LC and HC have at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 16 and 57, respectively. In some embodiments, this specification provides a bispecific antibody represented as D4, where LC and HC have the amino acid sequences shown in SEQ ID NOs: 16 and 57, respectively.

[0205] 2.6 variant This disclosure also considers other variants and equivalents 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 biological properties including, but not limited to, antibody specificity, heat resistance, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that changes in amino acids may affect post-translational modification processes of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

[0206] This specification further 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 embodiments. Functional variants of the heavy chain, light chain, VL, VH, or CDR used in the context of antibodies still allow for the retention of a significant portion (at least about 90%, 95% or more) of the functional properties (including affinity and / or specificity / selectivity, Fc inactivity, and PK parameters such as half-life, Tmax, and Cmax) of the “reference” and / or “parent” antibody. Such functional variants typically retain significant sequence identity with the parent antibody and / or have similar lengths to the heavy and light chains. Exemplary variants include those that differ primarily from the heavy and / or light chain, VH and / or VL, and / or CDR regions of the parent antibody sequence, for example, 10 of the variant substitutions, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1, may be substitutions of conservative amino acid residues.

[0207] In some embodiments, variants of the bispecific antibodies disclosed herein may retain the ability to bind to TNFα and IL23p19 to a similar, equal, or greater degree than that of the parent bispecific antibody. In some embodiments, the variants have 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 more homology in amino acid sequence with the parent antibody or antigen-binding fragment. In certain embodiments, variants of the bispecific antibodies disclosed herein include one or more conserved amino acid substitutions in the amino acid sequence of the parent bispecific antibody disclosed herein. Conserved amino acid substitutions are well known in the art and include amino acid substitutions in which one amino acid having specific physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties.

[0208] In some embodiments, the bispecific antibody variants disclosed herein include an amino acid sequence of a parent antibody having one or more non-conservative amino acid substitutions. In some embodiments, the bispecific antibody variants disclosed herein include an amino acid sequence of a parent-bound 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 the biological activity of one or more of the variants. In some 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, resulting in increased biological activity of the functional variant compared to the parent antibody.

[0209] In some embodiments, the mutant may have 1, 2, 3, 4, or 5 amino acid substitutions in the binding site CDR (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 located in their Fc region. In some embodiments, the modified antibody (e.g., the modified Fc region) results in altered effector function, thereby affecting the antibody's biological profile. For example, in some embodiments, deletion or inactivation of the constant region (by point mutation or other means) during circulation reduces Fc receptor binding of the modified antibody. In some embodiments, the modification of the constant region reduces the immunogenicity of the antibody. In some embodiments, the modification of the constant region increases the serum half-life of the antibody. In some embodiments, the modification of the constant region decreases the serum half-life of the antibody. In some embodiments, the modification of the constant region reduces or eliminates ADCC and / or complement-dependent cell-mediated cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acid substitutions in the human IgG1 Fc region having corresponding IgG2 or IgG4 residues reduce effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody has no effector function (e.g., an "effectorless" antibody). In some embodiments, the antibody has no ADCC activity and / or CDC activity. In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody has no effector function. In some embodiments, modification of the constant region increases or enhances the ADCC and / or CDC of the antibody. In some embodiments, the constant region is modified to remove a disulfide bond or oligosaccharide moiety. In some embodiments, the constant region is modified to add / substitute one or more amino acids to provide a binding site for one or more cytotoxins, oligosaccharides, or carbohydrates.

[0211] In some embodiments of the bispecific antibodies provided herein, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor. The Fc receptor may be a human Fc receptor. The Fc receptor may be an Fcγ receptor. The Fc receptor may be an activated Fc receptor. The Fc receptor may be an activated human Fcγ receptor such as human Fcγ RIIIa, Fcγ RI, or Fcγ RIIa. In some embodiments of the bispecific antibodies provided herein, the Fc domain includes one or more amino acid substitutions that reduce effector function. The effector function may be complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell 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, each of the two subunits of the Fc region has the same one or more amino acid substitutions. In one aspect, the one or more amino acid substitutions reduce the binding affinity of the Fc region to the Fc receptor. In another aspect, the one or more amino acid substitutions reduce the binding affinity of the Fc region to the Fc receptor by at least twofold, at least fivefold, or at least tenfold.

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

[0214] In some embodiments, the variant may involve adding an amino acid residue to the amino-terminus and / or carboxyl-terminus of the antibody. The length of the additional amino acid residue may range from 1 to 100 or more residues. In some embodiments, the variant includes an N-terminal methionyl residue. In some embodiments, the variant may be designed to be detectable and may include a detectable label and / or protein (e.g., a fluorescent tag or enzyme).

[0215] The mutant antibodies described herein can be produced using methods well known to those skilled in the art, including, but not limited to, site-directed mutagenesis, alanine scan mutagenesis, and PCR mutagenesis.

[0216] In some embodiments, the bispecific antibodies disclosed herein may be naturally or artificially chemically modified. In some embodiments, the bispecific antibodies are chemically modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protective / blocking groups, protease cleavage, and / or binding to cellular ligands or other proteins. Various chemical modifications may be carried out by known techniques. The bispecific antibodies provided herein may include one or more analogues of amino acids (e.g., including non-natural amino acids) and other modifications known in the art.

[0217] The bispecific antibodies of this disclosure can be analyzed for their physical, chemical, and / or biological properties by various methods well known in the art. In some embodiments, the ability of the bispecific antibodies provided herein to bind to human TNFα and / or human IL23p19 is tested. Binding tests include, but are not limited to, BLI, SPR (e.g., Biacore), ELISA, and FACS. Furthermore, the solubility, stability, heat resistance, viscosity, expression level, expression quality, and / or purification efficiency of the antibodies can be evaluated.

[0218] In some embodiments, the bispecific antibodies disclosed herein can be conjugated to detectable substances or molecules that enable the use of the drug for detection. Detectable substances include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase), prosthetic groups (e.g., biotin and flavin), fluorescent substances (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansilchloride, cyanine (Cy3), phycoerythrin), bioluminescent substances (e.g., luciferase), and radioactive substances (e.g., 212 Bi, 14 C, 57 Co, 51 Cr, 67 Cu, 18 F, 68 Ga, 67 Ga, 153 Gd, 159 Gd, 68 Enjoy, 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 This includes, but is not limited to, Zn, positron-emitting metals, and magnetic metal ions.

[0219] The anti-TNFα / IL23p19 bispecific antibodies disclosed herein may be conjugated to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the bispecific antibody immobilized for immunoassay is used. In some embodiments, the bispecific antibody immobilized for purification is used.

[0220] 3. Polynucleotides, vectors, and cells This specification 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 one polypeptide, such as the light chain or heavy chain of a bispecific antibody. In some embodiments, the polynucleotides provided herein encode multiple polypeptides. In some embodiments, the polynucleotides provided herein can, for example, encode the light chain and heavy chain of a bispecific antibody provided herein, respectively. Cistrons can be separated, for example, by 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 to mRNA, can directly recruit ribosomes without pre-scanning of the untranslated region of the mRNA by ribosomes. As understood in the art, a 2A element encodes a self-cleaving short peptide (about 20 amino acids) that provides a mechanism for subsequent separation of the target polypeptide produced in equimolar amounts. 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, this specification provides polynucleotides encoding LC1, HC1, LC2, HC2, or any combination thereof of the anti-TNFα / IL23p19 bispecific antibodies disclosed herein, having a CrossMab-KIH structure. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of the anti-TNFα / IL23p19 bispecific antibodies disclosed herein, having an IgG-ScFv structure, a DVD-Ig structure, or a SMAB structure.

[0222] As used herein, the term “encodes” and its grammatical equivalents refer to the inherent property of a particular nucleotide sequence of a polynucleotide or nucleic acid (e.g., a gene, cDNA, or mRNA) to function as a template for the synthesis of other polymers and macromolecules in a biological process having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties that result from this. Thus, if a protein is produced by the transcription and translation of the mRNA corresponding to the gene, the gene encodes the protein. Unless otherwise defined, “nucleotide sequences encoding an amino acid sequence” include all nucleotide sequences that encode the same amino acid sequence, including degenerate versions of each other. Nucleotide sequences encoding proteins and RNA may contain introns.

[0223] In some embodiments, this specification provides polynucleotides encoding LC1, HC1, LC2, HC2, or any combination thereof of a bispecific antibody represented by A1. In some embodiments, this specification provides a plurality of polynucleotides collectively encoding LC1, HC1, LC2, and HC2 of a bispecific antibody represented by A1. In some embodiments, this specification provides polynucleotides encoding LC1, HC1, LC2, HC2, or any combination thereof of a bispecific antibody represented by A2. In some embodiments, this specification provides a plurality of polynucleotides collectively encoding LC1, HC1, LC2, and HC2 of a bispecific antibody represented by A2. In some embodiments, this specification provides polynucleotides encoding LC1, HC1, LC2, HC2, or any combination thereof of a bispecific antibody represented by A3. In some embodiments, this specification provides a plurality of polynucleotides collectively encoding LC1, HC1, LC2, HC2 of a bispecific antibody represented by A3. In some embodiments, this specification provides polynucleotides encoding LC1, HC1, LC2, HC2, or any combination thereof of a bispecific antibody represented by A4. In some embodiments, this specification provides a plurality of polynucleotides that collectively encode LC1, HC1, LC2, and HC2 of a bispecific antibody represented by A4. In some embodiments, this specification provides polynucleotides that encode LC1, HC1, LC2, HC2, or any combination thereof of a bispecific antibody represented by A5. In some embodiments, this specification provides a plurality of polynucleotides that collectively encode LC1, HC1, LC2, and HC2 of a bispecific antibody represented by A5. In some embodiments, this specification provides polynucleotides that encode LC1, HC1, LC2, HC2, or any combination thereof of a bispecific antibody represented by A6. In some embodiments, this specification provides a plurality of polynucleotides that collectively encode LC1, HC1, LC2, and HC2 of a bispecific antibody represented by A6.In some embodiments, this specification provides polynucleotides encoding LC1, HC1, LC2, HC2, or any combination thereof of a bispecific antibody represented by A7. In some embodiments, this specification provides a plurality of polynucleotides collectively encoding LC1, HC1, LC2, and HC2 of a bispecific antibody represented by A7. In some embodiments, this specification provides polynucleotides encoding LC1, HC1, LC2, HC2, or any combination thereof of a bispecific antibody represented by A8. In some embodiments, this specification provides a plurality of polynucleotides collectively encoding LC1, HC1, LC2, and HC2 of a bispecific antibody represented by A8.

[0224] In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by B1. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by B1. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by B2. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by B2. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by B3. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by B3. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by B4. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by B4. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by B5. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC of a bispecific antibody represented by B5, respectively. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by B6. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC of a bispecific antibody represented by B6, respectively. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by B7. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC of a bispecific antibody represented by B7, respectively.In some embodiments, this specification provides polynucleotides encoding the LC, HC, or both of a bispecific antibody represented by B8. In some embodiments, this specification provides first and second polynucleotides encoding the LC and HC, respectively, of a bispecific antibody represented by B8.

[0225] In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by C1. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by C1. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by C2. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by C2. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by C3. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by C3. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by C4. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by C4. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by C5. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC of a bispecific antibody represented by C5, respectively. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by C6. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC of a bispecific antibody represented by C6, respectively. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by C7. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC of a bispecific antibody represented by C7, respectively.In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by C8. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by C8.

[0226] In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by D1. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by D1. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by D2. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by D2. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by D3. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by D3. In some embodiments, this specification provides polynucleotides encoding LC, HC, or both of a bispecific antibody represented by D4. In some embodiments, this specification provides first and second polynucleotides encoding LC and HC, respectively, of a bispecific antibody represented by D4.

[0227] The term "polynucleotide encoding a polypeptide" includes polynucleotides containing only the polypeptide coding sequence, and polynucleotides containing other coding sequences and / or non-coding sequences. The polynucleotides of the present invention may be in the form of RNA or DNA. The DNA may be cDNA, genomic DNA, or synthetic DNA, and may be double-stranded or single-stranded. The single-stranded DNA may be either a coding strand or a non-coding (antisense) strand. The polynucleotides of the present invention may be mRNA.

[0228] The present invention further provides a variant of the polynucleotide described herein, wherein the variant has a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the polynucleotide sequence encoding at least one polypeptide chain of the anti-TNFα / IL23p19 bispecific antibody described herein. As used herein, the phrase "a polynucleotide having a nucleotide sequence having at least about 95% identity with the polynucleotide sequence" means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations in each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence having at least 95% identity with the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These reference sequence mutations may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or at any position between those terminal positions, and may occur individually between nucleotides in the reference sequence, or scattered across one or more consecutive groups within the reference sequence.

[0229] The polynucleotide variant may include modifications in the coding region, the non-coding region, or both. In some embodiments, the polynucleotide variant includes modifications resulting in silent substitutions, additions, or deletions, but without altering the properties or activity of the encoded polypeptide. In some embodiments, the polynucleotide variant includes silent substitutions (due to degeneracy of the genetic code) that do not alter the amino acid sequence of the polypeptide. Polynucleotide variants may be generated for a variety of reasons, such as optimizing codon expression in a particular host (e.g., changing codons in human mRNA to codons preferred by a bacterial host such as E. coli). In some embodiments, the polynucleotide variant includes at least one silent mutation in the non-coding region or coding region of the sequence.

[0230] In some embodiments, polynucleotide variants are generated to modulate or alter the expression (or expression level) 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, polynucleotide variants increase the expression of the encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants decrease the expression of the encoded polypeptide compared to the parent polynucleotide sequence.

[0231] In some embodiments, the polynucleotide comprises a coding sequence of a polypeptide (e.g., an antibody) fused in the same reading frame as a polynucleotide (e.g., a leader sequence that functions as a secretory sequence to control polypeptide transport) that assists in the expression and secretion of the polypeptide from a host cell. The polypeptide comprises a leader sequence that is cleaved by the host cell to form a “mature form” of the polypeptide.

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

[0233] In some embodiments, the polynucleotides are isolated. In some embodiments, the polynucleotides are substantially pure.

[0234] In some embodiments, this specification provides vectors comprising polynucleotides provided herein. The term “vector” and its grammatical equivalents as used herein refer to a medium used to carry genetic material (e.g., polynucleotide sequences) that may be introduced into a host cell for replication and / or expression. Suitable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selectable sequences or markers that enable stable integration into the chromosomes of a host cell. Furthermore, the vectors may contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes may include those that provide resistance to antibiotics or toxins, compensate for nutritional deficiencies, or supply essential nutrients not present in the culture medium. Expression regulatory sequences may include constitutive or inductive promoters, transcriptional enhancers, transcriptional terminators, and the like, which are well known in the art. When expressing two or more polynucleotides collectively, the two polynucleotides may be inserted, for example, into a single expression vector or into separate expression vectors. In the case of single-vector expression, the encoded polynucleotide may be functionally ligated to a single common expression regulatory sequence, or it may be ligated to different expression regulatory sequences, such as a single inductive promoter and a single constitutive promoter. The introduction of the polynucleotide into host cells can be confirmed using methods well known in the art. Those skilled in the art will understand that the polynucleotide will be expressed in an amount sufficient to produce the target product, and further understand that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

[0235] In some embodiments, the vectors provided herein may be expression vectors. In some embodiments, the vectors provided herein contain a polynucleotide encoding at least one polypeptide chain of the anti-TNFα / IL23p19 bispecific antibody described herein. In some embodiments, the herein provides recombinant expression vectors that can be used to amplify and express a polynucleotide encoding at least one polypeptide chain of the anti-TNFα / IL23p19 bispecific antibody described herein. For example, the recombinant expression vector may be a replicable DNA construct containing a synthetic or cDNA-derived DNA fragment encoding at least one polypeptide chain of the anti-TNFα / IL23p19 bispecific antibody described herein, and operably ligated to a suitable transcription and / or translation regulatory element derived from a mammalian, microorganism, viral, or insect gene. In some embodiments, a viral vector is used. DNA regions are said to be "operably ligated" if they are functionally related to each other. For example, a promoter is functionally ligated to a coding sequence if it controls the transcription of the coding sequence. Or, a ribosome binding site is functionally ligated to a coding sequence if it is positioned to enable translation. In some embodiments, structural elements intended for use in a particular expression system include a leader sequence that enables extracellular secretion of the translated protein by the host cell. In some embodiments, when the recombinant protein is expressed without a leader sequence or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0236] Examples of vectors include plasmids, autonomous replication sequences, and transposable elements. Useful expression vectors for bacterial hosts include well-known bacterial plasmids such as E. coli plasmids containing pCR1, pBR322, pMB9, and their derivatives, and plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA phages. Further exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), and P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Examples of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST®, pLenti6 / V5-DEST®, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Exemplary transposon systems that can be stably integrated into genomes, such as Sleeping Beauty and PiggyBac, can be used (e.g., Ivics et al., Cell, 91 (4): 501-510 (1997); Cadinanos et al., (2007) Nucleic Acids Research. 35 (12): e87).

[0237] In some embodiments, the vector is an episomal vector or a vector maintained outside the chromosome. As used herein, the term “episome” refers to a vector that can replicate without being incorporated into the host’s chromosomal DNA and without being gradually lost from the dividing host cell, and also means that the early vector replicates outside the chromosome or episomally. The vector is engineered to contain a sequence encoding a DNA replication origin or “ori” derived from a lymphoid herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, particularly a replication origin of a lymphoid herpesvirus or gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphoid herpesvirus may be Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), herpesvirus thymili (HS), or Marek’s disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Typically, the host cell contains viral replication transactivator proteins that activate replication.

[0238] The "expression regulatory sequences," "regulatory factors," or "regulatory elements" present in an expression vector are the vector's untranslated regions, namely the origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Schein-Dalgano sequence or Kozak sequence), introns, polyadenylated sequences, and the 5' and 3' untranslated regions. These interact with host cell proteins to perform transcription and translation. These elements may differ in intensity and specificity. Depending on the vector system and host used, appropriate transcription and translation elements, including any number of ubiquitous and inducible promoters, can be used.

[0239] Exemplary universal expression regulatory sequences available for use in this disclosure include the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) promoter (e.g., early or late), the Moloney's mouse leukemia virus (MoMLV) LTR promoter, the Roussarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the vaccinia virus-derived H5, P7.5, and P11 promoters, and the elongation factor 1. This includes, but is not limited to, the 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 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and β-actin promoter.

[0240] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoids or estrogen receptors (inducible by treatment with the corresponding hormone), metallothione promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone regulatory system (Sirin et al, 2003, Gene, 323:67), cumic acid-inducible gene switches (WO 2002 / 088346), and tetracycline-dependent regulatory systems. The bispecific antibodies described herein can be produced by any method well known in the art, such as chemical synthesis and recombinant expression techniques. Unless otherwise specified, the implementation of the present invention utilizes prior art in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields in the art. These techniques are described in and in detail in the references cited herein. 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, Second Edition, Cold Spring Harbor Laboratory Press, Sambrook et al. NY, Ausubel et al.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, Second Edition, Oxford University Press, Lo (ed.) (2006) ANTIBODY ENGINEERING: You may also refer to METHODS AND PROTOCOLS (METHODS IN MOLECULAR BIOLOGY), Vol. 248, Humana Press, Inc., and each of the above references is incorporated herein by reference in its entirety.

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

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

[0243] Cells containing the vectors disclosed herein are also considered. In some embodiments, host cells containing the polynucleotides disclosed herein are also provided. In some embodiments, the host cells provided herein contain a vector or a plurality of vectors collectively containing polynucleotides encoding the polypeptide chain of the anti-TNFα / IL23p19 bispecific antibody described herein. In some embodiments, the host cells provided herein produce the anti-TNFα / IL23p19 bispecific antibody described herein.

[0244] Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovaries), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human fetal kidney) cell lines and their variants. Mammalian expression vectors may include non-transcriptional elements, including an origin of replication, a suitable promoter and enhancer linked to the expressed gene, other 5' or 3' adjacent non-transcriptional sequences, and 5' or 3' untranslated regions (e.g., required ribosome binding sites, polyadenylation sites, splice donor / acceptor sites, and transcription termination sequences). Recombinant protein expression in insect cell culture systems (e.g., baculoviruses) provides a powerful 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.

[0245] 4. Production Method This specification further provides methods for producing the anti-TNFα / IL23p19 bispecific antibody disclosed herein. In some embodiments, the bispecific antibody disclosed herein comprises one or more polypeptide chains, which can be produced separately or together. In some embodiments, the method provided herein produces at least one polypeptide chain of the bispecific antibody disclosed herein. In some embodiments, the method provided herein produces all polypeptide chains of the bispecific antibody disclosed herein.

[0246] The bispecific antibodies or polypeptides described herein can be produced and isolated using methods well known in the art. Polypeptides can be synthesized whole or in part using chemical methods (see, for example, Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, AK, Therapeutic Peptides And Proteins, Formulation, Processing And Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be carried out using various solid-phase techniques (see, for example, Roberge, Science 269:202 (1995); Merrifield, Methods. Enzymol. 289:3 (1997)), and automated synthesis can be carried out, for example, using an ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions. Peptides can also be synthesized using combinatorial methodologies. Synthetic residues and polypeptides can be synthesized using various procedures and methodologies well known in the art (see, for example, Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification (see, for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994)). Mutation, induction, substitution, and modification of peptide sequences can be carried out using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis.The peptide sequences, variants, fusions, and chimeras of the present invention, as well as their variants, derivatives, substitutions, and variants, derivatives, and variants thereof, can be produced by performing 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 SerA 317:415 (1986)) and other techniques on cloned DNA.

[0247] Various host expression vector systems can be used to recombinantly express one or more of the bispecific antibodies or their polypeptide chains described herein. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of an appropriate promoter. 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. Such host expression systems represent a medium that can generate and subsequently purify the coding sequences of the bispecific antibodies described herein, but also represent cells that, when transformed or transfected with an appropriate polynucleotide coding sequence, can in situ express the bispecific antibodies described herein. These include microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the sequences encoding the compounds described herein, and yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the sequences encoding the compounds described herein. This includes, but is not limited to, insect cell lines infected with a recombinant viral expression vector (e.g., baclovirus) containing a sequence encoding the compounds described herein, plant cell lines infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)), or plant cell lines transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing a sequence 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)), or C.6 cells (human retinal cells developed by Crucell) having a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., late adenovirus promoter, vaccinia virus 7.5K promoter).

[0248] In bacterial systems, many expression vectors can be advantageously selected depending on the intended use of the expressed protein. For example, when producing large quantities of such proteins, a vector that induces the expression of a high level of easily purifiable protein product is desirable for manufacturing the bispecific antibody pharmaceutical compositions described herein. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. (1983), EMBO J. 2: 1791-1794) and pIN vectors (Inouye et al. (1985), Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989), J. Biol. Chem. 24:5503-5509). pGEX vectors are also used to express polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such proteins are soluble and can be easily purified from lysed cells by adsorbing and binding them to a matrix of glutathione-agarose beads and eluting them in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST region.

[0249] Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Many virus-based expression systems can be used in mammalian host cells. Suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovaries), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human fetal kidney) cell lines and their mutants. Mammalian expression vectors may include a replication origin, appropriate promoters and enhancers linked to the expressed gene, other 5' or 3' adjacent non-transcriptional sequences, and non-transcriptional elements including 5' or 3' untranslated regions (such as required ribosome binding sites, polyadenylation sites, splice donor / acceptor sites, and transcription termination sequences). Recombinant protein expression in insect cell culture systems (e.g., baculoviruses) provides a powerful method for producing correctly folded, biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art. Autographa californica nuclear polyhedron disease virus (AcNPV) is used as a vector for expressing foreign genes.

[0250] Furthermore, host cell lines can be selected that regulate the expression of the inserted sequence or modify / process the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for protein function. For example, in some embodiments, the antibodies described herein may be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a polyprotein precursor) and require proteolytic cleavage by natural or recombinant cellular mechanisms to form separate polypeptides of the bispecific antibody described herein. Accordingly, this disclosure involves manipulating a nucleic acid sequence encoding a polyprotein precursor molecule containing the bispecific antibody polypeptide described herein, the nucleic acid sequence comprising a coding sequence capable of inducing post-translational cleavage of the polyprotein precursor. Post-translational cleavage of the polyprotein precursor generates the bispecific antibody polypeptide described herein. Posttranslational cleavage of precursor molecules containing polypeptides of the compounds described herein may occur in vivo (i.e., by innate or recombinant cellular systems / mechanisms within host cells, e.g., by Furin cleavage at appropriate sites) or in vitro (e.g., by culturing the polypeptide chain in a composition containing a protease or peptidase of known activity and / or conditions or reagents known to promote the desired proteolytic activity). The purification and modification of recombinant proteins are well known in the art, and the design of polyprotein precursors may include numerous embodiments readily understandable to skilled technicians. Any protease or peptidase known in the art may be used for the described modifications of the precursor molecules.

[0251] Various host cells possess characteristic and specific mechanisms for the post-translational processing or modification of proteins and gene products. By selecting an appropriate cell line or host system, the correct modification or processing of expressed exogenous proteins can be ensured. For this purpose, eukaryotic host cells with cellular mechanisms for appropriately handling the primary transcription, glycosylation, and phosphorylation of gene products can be used. Such 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] Stable expression is desirable for the long-term, high-yield production of recombinant proteins. For example, cell lines that stably express the compounds described herein can be constructed. Instead of using expression vectors containing viral replication origins, host cells can be transformed using DNA controlled by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selection markers. After introduction of exogenous DNA, the modified cells are grown in concentrated medium for 1-2 days, then switched to selective medium. The selection markers of the recombinant plasmid confer resistance to selection, allowing cells to stably incorporate the plasmid into their chromosomes, grow, and form a foci, which can then be cloned and expanded into a cell line. This method can be advantageously used to create cell lines that express the compounds described herein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that directly or indirectly interact with the compounds described herein.

[0253] Numerous selective systems may be used, including but not limited to the genes for 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), and can be used in tk-, hgprt-, or aprt- cells, respectively. Furthermore, antimetabolite resistance can be used as a basis for selecting the following genes: dhfr (Wigler et al. (1980) PNAS 77:3567-3570; O'Hare et al. (1981) PNAS, 78: 1527-1531) which confers resistance to methotrexate; gpt (Mulligan et al. (1981) PNAS, 78: 2072-2076) which confers resistance to mycophenolate; and neo (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), hygro (Santerre et al. (1984) Gene 30: 147-156) confers resistance to hygromycin.Methods commonly known in the field 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 levels of the bispecific antibodies or their polypeptide chains described herein can be increased by vector amplification (see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987) for a review). If the markers in the vector systems described herein are amplified, an increase in the level of the inhibitor present in the host cell culture system will increase the copy number of the marker gene. Since the amplified region is related to the nucleotide sequence of the target protein, the production of the target protein will also increase (Crouse et al. (1983) Mol. Cell. Biol. 3:257-266).

[0255] A host cell can co-transfect a plurality of expression vectors encoding the polypeptide chains of the bispecific antibodies described herein. The vectors may contain the same selection marker that allows for equal expression of all polypeptides. Alternatively, a single vector encoding two or more polypeptides can also be used. The coding sequences of the polypeptides of the compounds described herein may include cDNA or genomic DNA.

[0256] When the bispecific antibodies described herein or the polypeptides described herein are recombinantly expressed, they can be purified by any method well known in the art for the purification of polypeptides, polyproteins or antibodies (e.g., similar to antibody purification schemes based on antigen selectivity), e.g., chromatography (e.g., ion exchange, affinity, especially affinity for a specific antigen (if the compound contains an Fc domain (or a portion thereof), optionally after protein A selection), size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of polypeptides or antibodies.

[0257] This specification provides a method for producing the anti-TNFα / IL23p19 bispecific antibody described herein or the polypeptide chain of the bispecific antibody described herein, the method comprising obtaining a cell as described herein and expressing a polynucleotide as described herein in the cell. In some embodiments, the method further comprises isolating and purifying the bispecific antibody or polypeptide chain described herein.

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

[0259] The anti - TNFα / IL23p19 bispecific antibodies provided herein can be further tested for reactivity with the target antigen(s) by Western blotting. Other methods well - 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 a Gator system (Probe Life) or an Octet - 96 system (Sartorius AG), or surface plasmon resonance (SPR) analysis using a BIACORE™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

[0260] 5. Pharmaceutical Compositions This specification further provides pharmaceutical compositions comprising the anti-TNFα / IL23p19 bispecific antibody disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the bispecific antibody disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be used for the treatment of inflammatory or autoimmune diseases.

[0261] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refer to a material suitable for administering a drug to an individual together with an active agent without causing undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition. In some embodiments, the pharmaceutical compositions disclosed herein may include one or more buffers, preservatives, isotonic agents, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of buffers, preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. See REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 19th edition, 1995.

[0262] In some embodiments, the pharmaceutical compositions provided herein include the anti-TNFα / IL23p19 bispecific antibody provided herein. The anti-TNFα / IL23p19 bispecific antibody may be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein include 1 to 1000 mg / mL of the anti-TNFα / IL23p19 bispecific antibody provided herein. In some embodiments, the pharmaceutical compositions include 10 to 500 mg / mL, 10 to 400 mg / mL, 10 to 300 mg / mL, 10 to 200 mg / mL, 10 to 100 mg / mL, 20 to 100 mg / mL, or 50 to 100 mg / mL of the anti-TNFα / IL23p19 bispecific antibody provided herein. In some embodiments, the pharmaceutical compositions provided herein contain anti-TNFα / IL23p19 bispecific antibodies provided herein in doses of approximately 10 mg / mL, approximately 20 mg / mL, approximately 30 mg / mL, approximately 40 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 70 mg / mL, approximately 80 mg / mL, approximately 90 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 150 mg / mL, approximately 180 mg / mL, approximately 200 mg / mL, approximately 300 mg / mL, approximately 500 mg / mL, approximately 800 mg / mL, or approximately 1000 mg / mL. The dosage can be easily adjusted by those skilled in the art, for example, if the purity decreases, the dosage may need to be increased.

[0263] pharmaceutically acceptable carriers that can be used with the compositions provided herein include any physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. In some embodiments, the carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., anti-TNFα / IL23p19 bispecific antibody) may be coated with materials to protect the active ingredient from the action of acids and other natural conditions that may inactivate it.

[0264] This specification further provides pharmaceutical compositions or formulations that improve the stability of anti-TNFα / IL23p19 bispecific antibodies and enable long-term storage. In some embodiments, the pharmaceutical compositions or formulations disclosed herein comprise (a) the anti-TNFα / IL23p19 bispecific antibody disclosed herein, (b) a buffer, (c) a stabilizer, (d) a salt, (e) a volume extender, and / or (f) a surfactant. In some embodiments, the pharmaceutical compositions or formulations are stable for at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least five years or longer. In some embodiments, the pharmaceutical compositions or formulations are stable when stored at 4°C, 25°C, or 40°C.

[0265] A buffer useful in a pharmaceutical composition or formulation disclosed herein may be a weak acid or base used to maintain the acidity (pH) of the solution near a selected value after the addition of another acid or base. A suitable buffer can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties also depend 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) containing an isotonic agent and, optionally, a pH adjuster, such as an acid or base known in the art. In some embodiments, the buffer is L-histidine. In some 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 stabilize pharmaceuticals. Such agents can stabilize proteins in various 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, raftonose, or maltose; polyols such as glycerol, mannitol, or sorbitol; cyclodextrins; or destran of any type and molecular weight; or PEG. In some embodiments, the stabilizer is selected to maximize the stability of the antibody in the lyophilized formulation. In some embodiments, the stabilizer is sucrose and / or arginine.

[0267] Volume extenders can be added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby facilitating accurate measurement and handling. Common volume extenders include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0268] A surfactant is an amphiphilic substance having hydrophilic and hydrophobic groups. Surfactants may be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbate, and 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 may 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 physician or patient adds a solvent and / or diluent before use.

[0271] The pharmaceutical compositions disclosed herein may contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, e.g., ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, e.g., ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallic acid, α-tocopherol, etc.; and (3) metal chelating agents, e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0272] 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, and polyethylene glycol), 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 the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0273] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial presence can be reliably ensured by the sterilization procedures described above and by the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It is also desirable to include isotonic agents such as sugars and sodium chloride in the compositions. Furthermore, the absorption of the injectable agent can be extended by incorporating absorption-delaying agents such as aluminum monostearate or gelatin.

[0274] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injections or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. In some embodiments, this specification provides pharmaceutical compositions comprising anti-TNFα / IL23p19 bispecific antibodies or cells, which are suitable for topical administration.

[0275] Pharmaceutical compositions or formulations must typically be sterile and stable under manufacturing and storage conditions. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, maintaining the required particle size in the case of dispersions, or by the use of surfactants. Often, compositions may contain, for example, sugars, polyhydric alcohols such as mannitol and sorbitol, or isotonic agents such as sodium chloride. Extending the absorption of injectable compositions can be achieved by including absorption-delaying agents in the composition, for example, monostearate and gelatin.

[0276] Sterile injectable solutions can be prepared by adding the required amount of active compound to a suitable solvent, compounding it with one or a combination of the components listed above as needed, and then performing sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other necessary components listed herein. For sterile powders used to prepare sterile injectable solutions, the preparation methods include vacuum drying and refrigerated drying (freeze-drying), which can yield powders of the active ingredient and any additional desired components from a pre-sterile filtered solution.

[0277] The amount of active ingredient that can be combined with a carrier substance in the pharmaceutical compositions or formulations disclosed herein may vary. In some embodiments, the amount of active ingredient that can be combined with a carrier substance is the amount that produces a therapeutic effect. Generally, this amount is in the range of about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient combined with a pharmaceutically acceptable carrier, out of 100%.

[0278] The pharmaceutical compositions disclosed herein can be prepared using carriers that protect the active ingredient from rapid release, such as in controlled-release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for producing such formulations are patented or commonly known to those skilled in the art. For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0279] This specification further provides a kit for manufacturing a pharmaceutical composition comprising an anti-TNFα / IL23p19 bispecific antibody disclosed herein. In some embodiments, the kit comprises the anti-TNFα / IL23p19 bispecific antibody disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit may comprise the anti-TNFα / IL23p19 bispecific antibody disclosed herein for administration to a subject. In certain embodiments, the kit comprises instructions regarding the manufacture and / or administration of the anti-TNFα / IL23p19 bispecific antibody.

[0280] 6. Methods and Uses The anti-TNFα / IL23p19 bispecific antibody provided herein can be used in medicine. This specification further provides 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 anti-TNFα / IL23p19 bispecific antibody disclosed herein. This specification further provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-TNFα / IL23p19 bispecific antibody disclosed herein. This specification further provides a method for treating an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-TNFα / IL23p19 bispecific antibody disclosed herein. In some embodiments, the subject is 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] This specification provides the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein as a pharmaceutical product. This specification further provides the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein in the treatment of autoimmune diseases. This specification further provides the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein in the treatment of inflammatory diseases. This specification provides the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein in the manufacture of pharmaceuticals for the treatment of autoimmune diseases. This specification provides the use of the anti-TNFα / IL23p19 bispecific antibody disclosed herein in the manufacture of pharmaceuticals for the treatment of inflammatory diseases.

[0282] As used herein in relation to a disease or condition, or a subject having a disease or condition, the term “treat” and its grammatical equivalents refer to an action that suppresses, eliminates, reduces, and / or improves the symptoms, severity, 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 to be delivered a therapeutic agent or pharmaceutical composition into the body of a subject by the methods described herein or by other methods well known in the art. The therapeutic agent may be a compound, polypeptide, antibody, cell, or group of cells. The administration of a therapeutic agent or pharmaceutical composition includes prescribing a therapeutic agent or pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), and intraperitoneal (IP) administrations; transdermal dosage forms such as creams, jellies, powders, and patches; oral dosage forms; and inhaled powders, sprays, suspensions, and rectal suppositories.

[0284] As used herein, the terms “effective dose,” “therapeutic effective dose,” and their grammatical equivalents refer to the administration of a drug to a subject, either alone or as part of a pharmaceutical composition, and as a single dose or as part of a series, in an amount capable of producing any detectable positive effect on the symptoms, aspects, or characteristics of a disease, disorder, or condition when administered to the subject. The therapeutic effective dose can be determined by measuring the relevant physiological effect. The exact required dose will vary from subject to subject, depending on the subject’s age, weight, overall health, severity of the condition being treated, and the clinician’s judgment. The appropriate “effective dose” in each case can be determined by routine experimentation by those skilled in the art.

[0285] As used herein, the term “subject” refers to any animal (e.g., mammal) that is a recipient of a particular treatment, including but not limited to humans, non-human primates, dogs, cats, rodents, etc. Mammals include, but are not limited to, livestock, sports animals, pets, primates, horses, dogs, cats, mice, rats, etc. A human subject in need of treatment is a human subject who has, is at risk of, or is suspected of having a disease. A subject with a disease can be identified by routine medical examinations, such as physical examinations, clinical examinations, organ function tests, CT scans, and ultrasound examinations. A subject suspected of having such a disease may exhibit one or more symptoms of that disease. The signs and symptoms of diseases, such as autoimmune diseases and inflammatory diseases, are well known to those skilled in the art. A subject at risk of a disease means a subject who has one or more risk factors for that disease. A subject may be a person. A subject is a subject suffering from a particular disease or condition.

[0286] Non-exclusive examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, psoriasis, type 1 diabetes, systemic lupus erythematosus, transplant rejection, autoimmune thyroid disease (Hashimoto's disease), sarcoidosis, scleroderma, granulomatous angiitis, Crohn's disease, ulcerative colitis, Sjögren's disease, ankylosing spondylitis, psoriatic arthritis, polymyositis, polyarteritis nodosa, immune-mediated bullous skin diseases, Behçet'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-specific 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), Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac splenic dermatitis, chronic fatigue syndrome immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, scarring pemphigoid, cold agglutinin disease, CRESTO syndrome, Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, lupus discoid, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, and Hashimoto's disease. This includes, but is not limited to, thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, inflammatory myopathy, insulin-dependent diabetes mellitus (Type 1), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyendocrine syndrome, polymyalgia rheumatoid arthritis, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, Stiffman syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's 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, such as plaque psoriasis, hidradenitis suppurativa, palmoplantar pustulosis, pityriasis rubra pilaris, atopic dermatitis, systemic sclerosis, Takayasu's 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 psoriasis vulgaris. 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 dose of the anti-TNFα / IL23p19 bispecific antibody or pharmaceutical composition disclosed herein is administered to a subject who can benefit from a reduction in autoimmunity. The subject may have undesirable, uncontrolled, or excessive autoimmune activation. The subject is at risk of developing undesirable, uncontrolled, or excessive autoimmune activation. The actual dose of the active ingredient of the pharmaceutical composition described herein (i.e., the anti-TNFα / IL23p19 bispecific antibody disclosed herein) can be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, and that is not toxic to the patient. The selected dose depends 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 field.

[0290] The anti-TNFα / IL23p19 bispecific antibodies disclosed herein can be administered as sustained-release formulations, in which case the frequency of administration may be reduced. The dosage and frequency of administration will vary depending on the half-life of the anti-TNFα / IL23p19 bispecific antibody in the patient. In therapeutic use, relatively high doses at relatively short intervals may be required until disease progression is suppressed or halted, or until the patient shows partial or complete improvement in the symptoms of the disease.

[0291] The anti-TNFα / IL23p19 bispecific antibodies or pharmaceutical compositions provided herein can be administered to subjects by any method known in the art, including but not limited to pleural administration, intravenous administration, subcutaneous administration, intra-lymph node administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal administration, or other parenteral administration routes, such as administration by injection or infusion, or direct administration to the thymus. As used herein, the term “parenteral administration” means administration methods other than enteral and topical administration, and is usually by injection, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intra-articular, intra-articular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intracutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. 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 antibodies or pharmaceutical compositions provided herein can be administered using medical devices well known in the art. For example, in some embodiments, needleless subcutaneous injection devices such as those disclosed in U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556 may be used. Examples of well-known implants and modules described herein include U.S. Patent No. 4,487,603, an implantable microinfusion pump for administering drugs at a controlled rate; U.S. Patent No. 4,486,194, a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,447,233, a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,439,196, an osmotic drug delivery system with multiple chambers; and U.S. Patent No. 4,475,196, an osmotic drug delivery system. These patents are incorporated herein by reference. Many of these implants, delivery systems, and modules are well known to those skilled in the art.

[0293] In some embodiments, the anti-TNFα / IL23p19 bispecific antibody or pharmaceutical composition provided herein may be administered in conjunction with additional therapy. Additional therapy may be performed before, concurrently with, or after administration of the bispecific antibody or pharmaceutical composition described herein. Concomitant administration may include simultaneous administration of a single pharmaceutical formulation or separate formulations, or sequential administration in any order, but generally involves administration within a period that allows all active agents to exert their biological activity simultaneously. Those skilled in the art can easily determine, based on the needs of the subject being treated, an appropriate regimen for administering the pharmaceutical composition described herein in combination with additional therapy, including the timing and dosage of any additional agents used in the concomitant therapy.

[0294] 7. Experiment The examples shown below are for illustrative purposes only and are not intended to limit the invention unless otherwise noted. Therefore, the invention should not be construed as being limited in any way to the following embodiments, but rather as including any variations that become apparent as a result of the teachings provided herein.

[0295] In short, the results of the study described below demonstrate that the bispecific antibody binds to both human TNFα and human IL23p19 with high affinity and effectively neutralizes the biological activity of human TNFα and human IL23p19 in vitro and in vivo.

[0296] The reference antibodies used in these studies include adalimumab (light and heavy chain: SEQ ID NOs. 3 and 4, respectively), golimumab (light and heavy chain: SEQ ID NOs. 7 and 8, respectively), ozoralizumab (SEQ ID NOs. 9), guselkumab (light and heavy chain: SEQ ID NOs. 12 and 13, respectively), and tildrakizumab (light and heavy chain: SEQ ID NOs. 16 and 17, respectively).

[0297] 7.1 Example 1: Expression and purification of bispecific antibodies Methods: The bispecific antibodies described were expressed and purified as follows. Actively proliferating Expi CHO-S cells were inoculated into serum-free expression medium and cultured at 37°C in 8% CO2 in a shaker. Cells were carefully inspected for contamination and cell density was measured. Plasmids were mixed with transfection reagents and cultured at room temperature for 2 minutes. The mixture was added to Expi CHO-S cells and cultured. 18–22 hours after transfection, enhancers and feed were added to the cells. Cells were centrifuged and the supernatant was collected. The supernatant was filtered through a 0.22 μm microfiltration membrane and loaded onto a purification column. The eluted antibodies were transferred to a dialysis bag and dialyzed with PBS. Samples of bispecific antibodies were stored at 4°C for testing, and the remainder was frozen in liquid nitrogen and stored at -70°C.

[0298] 7.2 Example 2: Binding affinity of bispecific antibodies against TNFα and IL23p19 Methods: Running buffer (HBS-EP+) was prepared by diluting 1 volume of 10× stock 9-fold with deaerated MilliQ water. Regeneration buffer (10 mM glycine) was prepared by dissolving glycine in MilliQ water and adjusting the pH to 1.5–1.7. Measurements were performed at 25°C. Antibodies were injected as capture into Series S sensor tip protein A. Recombinant antigens (TNFα or IL23p19) at different concentrations were injected as the conjugation phase onto the surface of flow cells 1 and 2, followed by injection of running buffer as the dissociation phase. Affinity data were obtained using analysis software.

[0299] Results and Conclusion: As shown in Figure 2, the exemplary bispecific antibodies disclosed herein bound to both human TNFα and human IL23p19 with high affinity comparable to that of the reference antibody.

[0300] 7.3 Example 3: Simultaneous binding of TNFα and IL-23p19 Methods: The bispecific antibody was first captured on a protein A sensor chip, and then 100 nM human IL-23 was injected for 120 seconds to saturate the IL-23 binding site of the bispecific antibody. Next, human TNFα was injected at a series of concentrations to form the conjugation phase, followed by the injection of running buffer to form the dissociation phase. Only one direction was applied. Affinity data were obtained using analysis software.

[0301] Results and Conclusions: As shown in Figure 3, the exemplary bispecific antibodies disclosed herein bound simultaneously to human TNFα and human IL23p19. Surprisingly, the TNFα binding affinity of the bispecific antibodies pre-bound to IL23p19 was equivalent to the affinity for TNFα as a single antigen measured in Example 2. This indicates that dual therapeutic objectives can be achieved by targeting two antigens separately and simultaneously using these exemplary bispecific antibodies, and that these antibodies may serve as a safer and more effective therapeutic option than antibodies targeting a single antigen.

[0302] 7.4 Example 4: Inhibition of TNFα-induced NF-κB luciferase signaling in TNFR reporter cell lines Methods: The inhibitory activity of exemplary bispecific antibodies provided herein against the soluble TNFα signaling pathway was measured by a TNFα neutralization assay using a luciferase reporter cell line. This cell line was engineered to include an NF-κB response element upstream of the luciferase reporter gene. TNFα binding to cell surface TNFRs mediated activation of the NF-κB signaling cascade and promotion of luciferase reporter gene expression. As shown, samples of exemplary bispecific antibodies, gradient diluted, were pre-incubated with TNFα in 96-well plates at ambient temperature. After incubation, cells were transferred to wells in assay plates and cultured. TNFα neutralizing ability was determined by luciferase signaling.

[0303] Results and Conclusions: As shown in Figures 4A, 4B, and 4C, the exemplary bispecific antibodies disclosed herein effectively inhibited human TNFα-induced NF-κB signaling with high potency. As shown in the figures, the inhibitory potency of the exemplary bispecific antibodies provided herein was comparable to that observed with the reference antibody.

[0304] 7.5 Example 5: Inhibition of IL-23-induced STAT3 phosphorylated luciferase signaling in IL-23R reporter cell lines Methods: The inhibitory activity of exemplary bispecific antibodies provided herein against the IL-23 signaling pathway was measured by an IL-23 neutralization assay using a luciferase reporter cell line. When this reporter cell line was cultured with human IL-23, STAT3 was phosphorylated by IL-23R / JAK kinase, and such phosphorylation was measured using a commercially available luciferase reporter gene assay reagent. As shown, samples of exemplary bispecific antibodies, obtained by gradient dilution, were pre-incubated with IL-23 in a 96-well plate at ambient temperature. After culturing, cells were transferred to wells of an assay plate and cultured. IL-23 neutralizing ability was determined by luciferase signaling.

[0305] Results and Conclusions: As shown in Figures 5A, 5B, and 5C, the exemplary bispecific antibodies disclosed herein effectively inhibited human IL-23-induced STAT3 phosphorylation signaling with high potency. As shown in the figures, the inhibitory potency of the exemplary bispecific antibodies provided herein was comparable to that observed with the reference antibody.

[0306] 7.6 Example 6: Inhibition of apoptosis in TNFα-inducible U-937 cells Methods: U-937 is a promonocyte human myeloid leukemia cell line that spontaneously expresses the TNF receptor. TNFα induces caspase-dependent cell apoptosis in U-937 cells. Inhibition of TNFα-induced cell apoptosis is determined by measuring the activity of caspase 3 / 7. Bispecific antibodies diluted with a gradient were cultured with U-937 cells for 48 hours. After culturing, the luminescence signal was measured using the Caspase-Glo® 3 / 7 assay system.

[0307] Results and Conclusions: As shown in Figure 6, the exemplary bispecific antibodies disclosed herein effectively inhibited apoptosis in human TNFα-induced U937 cells. The inhibitory efficacy of the exemplary bispecific antibodies provided herein was comparable to that observed with the reference antibody.

[0308] 7.7 Example 7: Inhibition of TNFα-induced cytotoxicity in L929 Methods: L929 is a mouse fibroblast cell line that naturally expresses the TNF receptor. TNFα induces death in L929 cells through the over-formation of reactive oxygen species intermediates. Inhibition of cytotoxicity by a bispecific antibody is determined by measuring cell viability. Bispecific antibodies and TNF-α were pre-incubated in gradient dilutions and added to L929 cells. The mixture was cultured at 37°C / 5% CO2. After culturing, the level of cytotoxicity was determined by measuring the luminescence signal.

[0309] Results and Conclusions: As shown in Figure 7, the exemplary bispecific antibodies disclosed herein effectively inhibited cytotoxicity in human TNFα-induced L929 cells. The inhibitory efficacy of the exemplary bispecific antibodies provided herein was comparable to that observed with the reference antibody.

[0310] 7.8 Example 8: Inhibition of IL-23 and TNFα combination-induced IL-17 cytokine production in human PBMCs Methods: TNFα is known to promote IL-17 secretion from Th17 cells. Human PBMCs were treated with a cytokine mixture containing TNFα and IL-23, pre-incubated with a titrated bispecific antibody. Cell supernatants were collected and IL-17 concentrations were analyzed using a commercially available ELISA kit.

[0311] Results and Conclusions: As shown in Figure 8, the exemplary bispecific antibodies disclosed herein effectively inhibited human IL-17 cytokine production induced by the combination of TNFα and IL-23 in human PBMCs. The inhibitory efficacy of the exemplary bispecific antibodies provided herein was superior to that observed with the reference antibody.

[0312] 7.9 Example 9: Inhibition of human TNFα-induced mIL-6 production in mice Methods: Mice were injected with the bispecific antibodies provided herein and challenged with human TNFα to induce in vivo IL-6 production. After the challenge, serum was collected from whole blood at time points, and mouse IL-6 levels were analyzed using a commercially available ELISA assay kit.

[0313] Results and Conclusion: As shown in Figure 9, the exemplary bispecific antibodies disclosed herein significantly inhibited human TNFα-induced in vivo IL-6 production in mice.

[0314] 7.10 Example 10: Inhibition of mouse ear hyperplasia by human IL-23 Methods: Mice were injected with bispecific antibodies and then intradermally administered human IL-23 to induce ear hyperplasia. Ear thickness, cytokine levels in ear tissue, and ear histopathological scores were evaluated.

[0315] Results and Conclusions: As shown in Figure 10, the exemplary bispecific antibodies disclosed herein significantly improved human IL-23-induced ear thickness, clinical PASI score, and histopathology in a mouse model of ear hyperplasia. The inhibitory efficacy of the bispecific antibodies provided herein was comparable to that observed with the reference antibody.

[0316] Thus, compared to monospecific antibodies targeting TNFα or anti-IL23, or combinations thereof, the bispecific antibodies provided herein offer the advantages of additional and / or synergistic therapeutic effects, comparable safety, cost reduction, and improved convenience, safety, and patient compliance as monotherapy.

[0317] Citation of electronically submitted sequence listings This application incorporates, by reference, an array listing file "[P22406692C].SEQ.XML" created on September 18, 2022, with a size of 142,259 bytes. [Brief explanation of the drawing]

[0318] [Figure 1A] The "CrossMab-KIH" structure is composed of four distinct polypeptides: a first light chain (LC1), a first heavy chain (HC1), a second light chain (LC2), and a second heavy chain (HC2), with the following composition: LC1: (N)-VL1-CH1-(C) HC1: (N)-VH1-CL region-Fc region (Knob)-(C) LC2: (N)-VL2-CL region-(C) HC2: (N)-VH2-CH1-Fc region (Hole)-(C). [Figure 1B]The "IgG-ScFv" structure contains two pairs of identical light chains (LC) and heavy chains (HC), with the following configuration: LC: (N)-VL1-CL region-(C) HC: (N)-VH1-CH region-VH2-VL2-(C). [Figure 1C] It has a "DVD-Ig" structure, which contains two pairs of identical light chains (LC) and heavy chains (HC), and its composition is as follows: LC: (N)-VL1-VL2-CL region-(C) HC: (N)-VH1-VH2-CH region-(C). [Figure 1D] The structure is "SMAB-VHH," which contains two pairs of identical light chains (LC) and heavy chains (HC), and its composition is as follows: LC: (N)-VL1-CL region-(C) HC: (N)-VHH-VH-CH region-(C) or (N)-VH-CH region-VHH-(C). [Figure 2] This chart summarizes the binding affinity of exemplary anti-TNFα / IL23p19 bispecific antibodies to TNFα and IL23p19, respectively. [Figure 3] This chart illustrates how an exemplary anti-TNFα / IL23p19 bispecific antibody simultaneously binds to two antigens, namely human TNFα and IL23p19. [Figure 4A] This graph illustrates the effective inhibition of TNFα-induced NFκB signaling by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 4B] This graph illustrates the effective inhibition of TNFα-induced NFκB signaling by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 4C] This graph illustrates the effective inhibition of TNFα-induced NFκB signaling by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 5A] This graph illustrates the effective inhibition of IL-23-induced STAT3 phosphorylation by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 5B]This graph illustrates the effective inhibition of IL-23-induced STAT3 phosphorylation by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 5C] This graph illustrates the effective inhibition of IL-23-induced STAT3 phosphorylation by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 6] This graph illustrates the effective inhibition of TNFα-induced U937 cell apoptosis by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 7] This graph illustrates the effective inhibition of cytotoxicity in TNFα-inducible L929 cells by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 8] This graph shows the effective inhibition of IL-23-induced human IL-17 cytokine production and the TNFα combination in human PBMCs by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 9] This graph illustrates the effective inhibition of human TNFα-induced mIL-6 production in mice by an exemplary anti-TNFα / IL23p19 bispecific antibody. [Figure 10] This graph illustrates the effective inhibition of human IL-23-induced ear hyperplasia in mice by an exemplary anti-TNFα / IL23p19 bispecific antibody, where ear thickness, clinical PASI score, and ear histopathology score are evaluated.

Claims

1. (1) A light chain (LC) comprising a first light chain variable domain (VL1) and a CL region, and (2) comprising a heavy chain (HC) containing 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), Here, the VL1 / VH1 pair and the VL2 / VH2 pair are bispecific antibodies that specifically bind to human TNFα and human IL23p19, respectively, or specifically bind to human IL23p19 and human TNFα, respectively, specifically binding to the P19 subunit of human TNFα and human IL23 (IL23 p19).

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

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

4. VL1 and VH1 have (2) the amino acid sequences shown in SEQ ID NOs. 5 and 6, respectively, or (1) SEQ ID NOs. 1 and 2, respectively, or VL2 and VH2 have (3) the amino acid sequences shown in SEQ ID NOs. 93 and 94, respectively, or (1) SEQ ID NOs. 10 and 11, respectively, or (2) SEQ ID NOs. 14 and 15, respectively. Alternatively, VL1, VH1, VL2, and VH2 have the amino acid sequences shown in (6) SEQ ID NOs. 5, 6, 93, and 94, respectively, or (1) SEQ ID NOs. 1, 2, 10, and 11, respectively, (2) SEQ ID NOs. 1, 2, 14, and 15, respectively, (3) SEQ ID NOs. 1, 2, 93, and 94, respectively, (4) SEQ ID NOs. 5, 6, 10, and 11, respectively, or (5) SEQ ID NOs. 5, 6, 14, and 15, respectively. The bispecific antibody according to claim 3.

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

6. The CH region includes modification, The aforementioned modification increases the serum half-life of the bispecific antibody, or The bispecific antibody according to claim 5, wherein the modifications are a substitution to tyrosine at amino acid residue 252, a substitution to threonine at amino acid residue 254, and a substitution to glutamic acid at amino acid residue 256, which are numbered according to the EU index.

7. A polynucleotide encoding LC, HC, or both LC and HC of the bispecific antibody described in claim 1.

8. A vector comprising the polynucleotide described in claim 7.

9. (a') a polynucleotide encoding both LC and HC, or (b') comprising a first polynucleotide encoding LC and a second polynucleotide encoding HC, A cell in which the polynucleotide in (a'), the first nucleotide and the second nucleotide in (b') are as defined in claim 7.

10. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 6, a polynucleotide according to claim 7 and / or a cell according to claim 9, and optionally a pharmaceutically acceptable carrier.

11. It is used to alleviate TNFα and / or IL23p19-related autoimmunity or inflammation in subjects, and to treat autoimmune diseases or inflammatory diseases. Preferably, the subject has an autoimmune disease or an inflammatory disease. A bispecific antibody according to any one of claims 1 to 6, a polynucleotide according to claim 7, and / or a cell according to claim 9.

12. Used to alleviate TNFα and / or IL23p19-related autoimmunity or inflammation in a subject, to treat autoimmune diseases or inflammatory diseases, Preferably, the subject has an autoimmune disease or an inflammatory disease. The pharmaceutical composition according to claim 10.

13. A method for producing a bispecific antibody that specifically binds to human TNFα and human IL23p19 by expressing the polynucleotide or a plurality of polynucleotides in the cells described in claim 9.

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