IL-23 binding molecules and uses thereof

Abstract

The present application provides anti-IL-23 single-domain antibodies with higher binding activity. The anti-IL-23 single-domain antibodies of the present application have human and monkey species cross-binding activity, and can block the binding of IL-23 to IL-23R, inhibit the IL-23 / IL-23R pathway, and thus have the development potential as therapeutic drugs for IL-23-mediated immunological and inflammatory diseases.

Description

Technical Field

[0001] This invention belongs to the field of antibody drugs, specifically relating to an IL-23 binding molecule and its uses. Background Technology

[0002] Interleukin-23 (IL-23) is an important cytokine belonging to the IL-12 family and plays a crucial role in the immune system. IL-23 exists as a heterodimer formed by two subunits (p19 and p40 subunits) linked by disulfide bonds. Its domains typically include an N-terminal signal peptide, a β-sheet structure, and an α-helix structure, which, upon binding, form a specific stereoconformation that allows it to bind to the IL-23 receptor (IL-23R) on the cell surface. IL-23 is mainly produced by macrophages and dendritic cells. Its main biological functions in the immune response include: (1) promoting the differentiation and survival of Th17 cells: IL-23 can activate Naive T cells to differentiate into Th17 cells, which play an important role in autoimmune diseases and inflammation; (2) enhancing the production of cytokines: IL-23 stimulates Th17 cells and other immune cells to release a variety of cytokines, such as IL-17 and IL-22, thereby regulating the inflammatory response; (3) supporting chronic inflammation: In a variety of chronic inflammatory diseases (such as psoriasis, Crohn's disease, ankylosing spondylitis, etc.), IL-23 shows the effect of promoting pathological inflammation; (4) enhancing the activity of NK cells and cytotoxic T cells: IL-23 may also promote anti-tumor immunity and anti-infection response by stimulating the activation of NK cells and CD8+ T cells.

[0003] Due to its role in maintaining Th17 cell function, IL-23 is associated with a variety of immune and inflammatory diseases. Besides inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, IL-23 also plays a role in diseases such as psoriasis, rheumatoid arthritis, and multiple sclerosis. Under these pathological conditions, IL-23 promotes the maintenance of inflammatory responses, leading to tissue damage and disease progression.

[0004] Drugs targeting IL-23 can be used to prevent and treat IL-23-related immune and inflammatory diseases. However, the reported single-domain antibodies targeting the p19 subunit of IL-23 (IL-23p19) have lower activity than marketed monoclonal antibodies. Therefore, the development of highly active anti-IL-23 single-domain antibodies is of great value. Summary of the Invention

[0005] The purpose of this invention is to provide a single-domain anti-IL-23 antibody with higher activity and its antigen-binding fragment, as well as the application of the single-domain anti-IL-23 antibody and its antigen-binding fragment in disease diagnosis and treatment.

[0006] In a first aspect of the invention, an anti-IL-23 single-domain antibody or an antigen-binding fragment thereof is provided, wherein the VHH chain of the anti-IL-23 single-domain antibody or the antigen-binding fragment thereof comprises a complementarity-determining region (CDR) selected from the group consisting of:

[0007] (Z1) CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4; or

[0008] (Z2) CDR1 shown in SEQ ID NO:20, CDR2 shown in SEQ ID NO:21 and CDR3 shown in SEQ ID NO:22.

[0009] In another preferred embodiment, any of the amino acid sequences described above further includes a derivative sequence which has been optionally added, deleted, modified and / or substituted with at least one amino acid and is capable of retaining IL-23 binding affinity.

[0010] In another preferred embodiment, the VHH chain further includes a frame region (FR).

[0011] In another preferred embodiment, the frame region (FR) includes camel-derived FR and human-derived FR.

[0012] In another preferred embodiment, the frame region (FR) includes FR1, FR2, FR3 and FR4.

[0013] In another preferred embodiment, the VHH chain comprises the following FRs:

[0014] (1) FR1 shown in SEQ ID NO:5, FR2 shown in SEQ ID NO:6, FR3 shown in SEQ ID NO:7 and FR4 shown in SEQ ID NO:8;

[0015] (2) FR1 shown in SEQ ID NO:12, FR2 shown in SEQ ID NO:13, FR3 shown in SEQ ID NO:15 and FR4 shown in SEQ ID NO:18;

[0016] (3) FR1 shown in SEQ ID NO:12, FR2 shown in SEQ ID NO:13, FR3 shown in SEQ ID NO:16 and FR4 shown in SEQ ID NO:18; or

[0017] (4) FR1 shown in SEQ ID NO:12, FR2 shown in SEQ ID NO:14, FR3 shown in SEQ ID NO:17 and FR4 shown in SEQ ID NO:18.

[0018] In another preferred embodiment, the VHH chain comprises the following FRs:

[0019] (1) FR1 shown in SEQ ID NO:23, FR2 shown in SEQ ID NO:24, FR3 shown in SEQ ID NO:25 and FR4 shown in SEQ ID NO:8; or

[0020] (2) FR1 shown in SEQ ID NO:27, FR2 shown in SEQ ID NO:24, FR3 shown in SEQ ID NO:25 and FR4 shown in SEQ ID NO:18.

[0021] In another preferred embodiment, the VHH chain of the anti-IL-23 single-domain antibody or its antigen-binding fragment has an amino acid sequence that is at least 80% (preferably at least 85%, more preferably at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the amino acid sequence shown in any one of SEQ ID NO:1, 9-11.

[0022] In another preferred embodiment, the VHH chain of the anti-IL-23 single-domain antibody or its antigen-binding fragment has an amino acid sequence that is at least 80% (preferably at least 85%, more preferably at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO:19 or 26.

[0023] In another preferred embodiment, the VHH chain of the anti-IL-23 single-domain antibody or its antigen-binding fragment has an amino acid sequence as shown in any one of SEQ ID NO: 1, 9-11.

[0024] In another preferred embodiment, the VHH chain of the anti-IL-23 single-domain antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO: 19 or 26.

[0025] In another preferred embodiment, the anti-IL-23 single-domain antibody includes a camel-derived single-domain antibody, a chimeric single-domain antibody, or a humanized single-domain antibody.

[0026] In another preferred embodiment, the anti-IL-23 single-domain antibody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0027] In another preferred embodiment, the anti-IL-23 single-domain antibody is a bivalent, preferably a heterodivalent.

[0028] In another preferred embodiment, the heterodimer comprises two VHH chains with different sequences, and the two VHH chains are connected by a linker peptide; preferably, the linker peptide is selected from (GS)n, (GGS)n, (GGGS)n, and (GGGGS)n, where n is a positive integer from 1 to 5.

[0029] In another preferred embodiment, the heterodivalent comprises a VHH chain having an amino acid sequence as shown in any one of SEQ ID NO:1, 9-11, and a VHH chain having an amino acid sequence as shown in SEQ ID NO:19 or 26.

[0030] In another preferred embodiment, the heterodivalent comprises a VHH chain having an amino acid sequence as shown in any one of SEQ ID NO:9-11, and a VHH chain having an amino acid sequence as shown in SEQ ID NO:26.

[0031] In another preferred embodiment, the heterodivalent has an amino acid sequence as shown in any one of SEQ ID NO:28-30.

[0032] In another preferred embodiment, the anti-IL-23 single-domain antibody or its antigen-binding fragment specifically binds to human IL-23 and cynomolgus monkey IL-23.

[0033] In another preferred embodiment, the anti-IL-23 single-domain antibody or its antigen-binding fragment is a blocking antibody that blocks the binding of IL-23 to IL-23R.

[0034] In another preferred embodiment, the anti-IL-23 single-domain antibody or its antigen-binding fragment specifically binds to the p19 subunit of IL-23.

[0035] In a second aspect of the invention, a fusion protein is provided, the structure of which from the N-terminus to the C-terminus is shown in formula Ia or Ib:

[0036] ALB(Ia);

[0037] BLA(Ib);

[0038] in,

[0039] A is an anti-IL-23 single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0040] B is the Fc fragment of IgG; and

[0041] L represents no or flexible joints.

[0042] In another preferred embodiment, the flexible connector is a peptide connector.

[0043] In another preferred embodiment, the peptide linker has 1-50 amino acids, more preferably 1-20 amino acids.

[0044] In another preferred embodiment, the peptide linker is selected from (GS)n, (GGS)n, (GGGS)n, and (GGGGS)n, where n is a positive integer from 1 to 5.

[0045] In another preferred embodiment, the peptide linker has a (GGGGS)n structure, where n is a positive integer from 1 to 5.

[0046] In another preferred embodiment, the Fc fragment of the IgG comprises the Fc fragment of human IgG.

[0047] In another preferred embodiment, the Fc fragment of the IgG is selected from the group consisting of the Fc fragments of IgG1, IgG2, IgG3, IgG4, or combinations thereof.

[0048] In a third aspect of the invention, a multispecific antibody is provided, the multispecific antibody comprising an anti-IL-23 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, or a fusion protein as described in the second aspect of the invention.

[0049] In another preferred embodiment, the multispecific antibody includes bispecific antibodies, trispecific antibodies, etc.

[0050] In another preferred embodiment, the multispecific antibody is a bispecific antibody.

[0051] In another preferred embodiment, the bispecific antibody comprises an anti-IL-23 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, and an anti-TNF-like ligand 1A (TL1A) single-domain antibody.

[0052] In another preferred embodiment, the anti-TL1A single-domain antibody comprises a VHH chain with an amino acid sequence as shown in SEQ ID NO:35.

[0053] In another preferred embodiment, the bispecific antibody comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other.

[0054] In another preferred embodiment, Fc1 has a protruding structure (knob) according to the knife-in-hole technique, and Fc2 has a hole structure (hole) according to the knife-in-hole technique.

[0055] In another preferred embodiment, Fc1 comprises the amino acid sequence shown in SEQ ID NO:31, and Fc2 comprises the amino acid sequence shown in SEQ ID NO:32.

[0056] In another preferred embodiment, Fc1 comprises an amino acid sequence as shown in SEQ ID NO:33, and Fc2 comprises an amino acid sequence as shown in SEQ ID NO:34.

[0057] In another preferred embodiment, the bispecific antibody comprises two peptide chains as shown in formulas (IIa) and (IIb):

[0058] C1-L1-Fc1(IIa)

[0059] C2-L2-Fc2(IIb)

[0060] Where "-" represents a peptide bond;

[0061] L1 and L2 are each independently a component without a connector;

[0062] C1 is an anti-IL-23 single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention, and C2 is an anti-TL1A single-domain antibody; or C1 is an anti-TL1A single-domain antibody, and C2 is an anti-IL-23 single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention; and

[0063] Fc1 is the first subunit of the Fc region of the antibody, and Fc2 is the second subunit of the Fc region of the antibody.

[0064] In another preferred embodiment, in the bispecific antibody, the amino acid sequence of the anti-IL-23 single-domain antibody is as shown in any one of SEQ ID NO:28-30, preferably as shown in SEQ ID NO:29 or 30; and

[0065] The amino acid sequence of the anti-TL1A single-domain antibody is shown in SEQ ID NO:35.

[0066] In a fourth aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0067] (i) an anti-IL-23 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0068] (ii) Optional tag sequences to assist in expression and / or purification.

[0069] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, and a His tag.

[0070] In a fifth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: an anti-IL-23 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a multispecific antibody as described in the third aspect of the invention, or a recombinant protein as described in the fourth aspect of the invention.

[0071] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0072] In a sixth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the fifth aspect of the invention.

[0073] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0074] In another preferred embodiment, the expression vector includes a viral vector, such as adenovirus, AAV virus, retrovirus, lentivirus, or a combination thereof.

[0075] In a seventh aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the sixth aspect of the invention, or having a genome integrated with polynucleotides as described in the fifth aspect of the invention.

[0076] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0077] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0078] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0079] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0080] In another preferred embodiment, the eukaryotic cells shown are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0081] In another preferred embodiment, the host cell is preferably a mammalian cell, more preferably a HEK293 cell, CHO cell, BHK cell, NSO cell, or COS cell.

[0082] In an eighth aspect of the invention, a method is provided for generating an anti-IL-23 single-domain antibody or its antigen-binding fragment, or its fusion protein, the multispecific antibody described in the third aspect of the invention, or the recombinant protein described in the fourth aspect of the invention, comprising the steps of:

[0083] (a) Under conditions suitable for protein expression, host cells as described in the seventh aspect of the present invention are cultured to obtain a culture containing the anti-IL-23 single-domain antibody or its antigen-binding fragment or its fusion protein, the multispecific antibody, and the recombinant protein;

[0084] (b) Isolating or recovering from the culture the anti-IL-23 single-domain antibody or its antigen-binding fragment, or its fusion protein, the multispecific antibody, or the recombinant protein; and

[0085] In another preferred embodiment, the method further includes the step of: (c) purifying and / or modifying the anti-IL-23 single-domain antibody or its antigen-binding fragment, or its fusion protein, multispecific antibody, or recombinant protein obtained in step (b).

[0086] In a ninth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0087] (i) an anti-IL-23 single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention; and

[0088] (ii) Pharmaceutically acceptable carriers.

[0089] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0090] In another preferred embodiment, the pharmaceutical composition is used to prepare for the prevention and / or treatment of IL-23-mediated immune and inflammatory diseases.

[0091] In another preferred embodiment, the IL-23-mediated immune and inflammatory diseases include, but are not limited to: inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; psoriasis, rheumatoid arthritis, palmoplantar pustulosis, psoriatic arthritis, hidradenitis suppurativa, pityriasis rubra pilaris, giant cell arteritis, pyoderma gangrenosa, lupus nephritis, familial benign pemphigus, metabolic-associated fatty liver disease, ankylosing spondylitis, non-radiological axial spondyloarthritis, vitiligo, asthma, atopic dermatitis, and multiple sclerosis.

[0092] In another preferred embodiment, the pharmaceutical composition may also contain other drugs for the prevention and / or treatment of immune and inflammatory diseases.

[0093] In a tenth aspect of the invention, an IL-23 inhibitor is provided, the inhibitor comprising an anti-IL-23 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a multispecific antibody as described in the third aspect of the invention, or a recombinant protein as described in the fourth aspect of the invention.

[0094] In another preferred embodiment, the inhibitor inhibits the activity of IL-23 by blocking the binding of IL-23 to IL-23R.

[0095] In an eleventh aspect of the present invention, the use of an anti-IL-23 single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention, for the preparation of:

[0096] (a) Medications used to prevent and / or treat IL-23-mediated diseases or conditions; or

[0097] (b) Detection reagents, test plates or test kits for detecting IL-23 molecules.

[0098] In another preferred embodiment, the IL-23-mediated diseases or conditions include immune and inflammatory diseases, including but not limited to: inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; psoriasis, rheumatoid arthritis, palmoplantar pustulosis, psoriatic arthritis, hidradenitis suppurativa, pityriasis rubra pilaris, giant cell arteritis, pyoderma gangrenosa, lupus nephritis, familial benign pemphigus, metabolic-associated fatty liver disease, ankylosing spondylitis, non-radiological axial spondyloarthritis, vitiligo, asthma, atopic dermatitis, and multiple sclerosis.

[0099] In another preferred embodiment, the detection includes flow cytometry, immunofluorescence assay, and ELISA.

[0100] In another preferred embodiment, the use is diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic.

[0101] In a twelfth aspect of the present invention, a method for detecting IL-23 protein in a sample is provided, the method comprising the steps of:

[0102] (1) Contact the sample with an anti-IL-23 single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention.

[0103] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of IL-23 protein in the sample.

[0104] In another preferred embodiment, the aspect is an in vitro method.

[0105] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0106] In a thirteenth aspect of the invention, a method for treating IL-23-mediated diseases or conditions is provided, the method comprising administering to a subject in need an anti-IL-23 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a multispecific antibody as described in the third aspect of the invention, a recombinant protein as described in the fourth aspect of the invention, or a pharmaceutical composition as described in the ninth aspect of the invention.

[0107] In another preferred embodiment, the object includes mammals, such as humans.

[0108] In another preferred embodiment, IL-23 is abnormally expressed in the subject's body.

[0109] In another preferred embodiment, the IL-23-mediated diseases or conditions include immune and inflammatory diseases, including but not limited to: inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; psoriasis, rheumatoid arthritis, palmoplantar pustulosis, psoriatic arthritis, hidradenitis suppurativa, pityriasis rubra pilaris, giant cell arteritis, pyoderma gangrenosa, lupus nephritis, familial benign pemphigus, metabolic-associated fatty liver disease, ankylosing spondylitis, non-radiological axial spondyloarthritis, vitiligo, asthma, atopic dermatitis, and multiple sclerosis.

[0110] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0111] Figure 1 The results are obtained by ELISA detection of the binding activity of the candidate IL-23p19 single-domain antibody to human IL-23.

[0112] Figure 2 The results are obtained by ELISA detection of the binding activity of the candidate IL-23p19 single-domain antibody to cynomolgus monkey IL-23.

[0113] Figure 3 The results are obtained by reporter gene assay to detect the inhibitory activity of candidate IL-23p19 single-domain antibody against the IL-23 / IL-23R pathway induced by human IL-23.

[0114] Figure 4A 4B and 4C are the results of reporter gene assay for detecting the inhibitory activity of humanized heterologous bivalent IL-23p19 single-domain antibody on the IL-23 / IL-23R pathway induced by human IL-23.

[0115] Figure 5 The results are obtained by reporter gene assay to detect the inhibitory activity of humanized heterologous bivalent IL-23p19 single-domain antibody against the IL-23 / IL-23R pathway induced by IL-23 in cynomolgus monkeys.

[0116] Figure 6 This is the result of Frotebio's detection of the cross-activity between the candidate single-domain antibody and natural IL-12.

[0117] Figure 7 This is the result of Frotebio's detection of the cross-activity between the candidate single-domain antibody and linearized IL-12.

[0118] Figure 8 The results are obtained by ELISA to identify the activity of humanized heterobivalent IL-23p19 single-domain antibody in inhibiting the release of IL-17 from the spleen of mice induced by human IL-23. Detailed Implementation

[0119] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly obtained two anti-IL-23 single-domain antibodies with higher activity. These anti-IL-23 single-domain antibodies not only bind to human IL-23 with high affinity but also to cynomolgus monkey IL-23, facilitating animal experimental studies. Furthermore, one of the single-domain antibodies is a blocking antibody, capable of blocking the binding of IL-23 to IL-23R, inhibiting the IL-23 / IL-23R pathway, and thus suppressing IL-23-mediated biological functions. The bivalent formed by tandemly combining the two anti-IL-23 single-domain antibodies of this invention exhibits better IL-23 binding and blocking activity compared to existing commercially available IL-23 monoclonal antibodies.

[0120] Based on this, the present invention was completed.

[0121] the term

[0122] To better understand this invention, the following terms are defined.

[0123] Unless the context clearly indicates otherwise, the term “about” includes values ​​within the standard deviation range of the stated value.

[0124] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0125] In this invention, "subject" or "patient" refers to an animal, including human patients requiring treatment for diseases mediated by the IL-23 pathway. In some aspects, the invention can also be applied in veterinary practice to any mammal or other animal requiring such IL-23-targeted therapy. This may include, for example, non-human primates, dogs, felines, pigs, horses, and any other animals requiring IL-23 treatment.

[0126] As used herein, the terms "single-domain antibody of the present invention", "IL-23 single-domain antibody of the present invention", "anti-IL-23 single-domain antibody of the present invention", "anti-IL-23 single-domain antibody", "IL-23 single-domain antibody", "anti-IL-23 nanobody", and "IL-23 nanobody" have the same meaning and can be used interchangeably. They all refer to the nanobodies that specifically recognize and bind to the IL-23 protein, especially the p19 subunit of the IL-23 protein, screened and prepared by the present invention.

[0127] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0128] As used herein, the terms "nanobody" and "single-domain antibody (sdAb)" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of the antibody heavy chain to construct a single-domain antibody consisting of only one heavy chain variable region (VHH). This is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0129] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0130] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0131] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0132] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to the IL-23 protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0133] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., antibody-drug conjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0134] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called complementarity-determining regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0135] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0136] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0137] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0138] The term "antibody of the present invention" refers to a polypeptide having IL-23 binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0139] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0140] The present invention also provides other polypeptides, such as fusion proteins comprising a single-domain antibody or a fragment thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the single-domain antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0141] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0142] Table A

[0143]

[0144]

[0145] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0146] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0147] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0148] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0149] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0150] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0151] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0152] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0153] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0154] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0155] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0156] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0157] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0158] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0159] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0160] Anti-IL-23 single-domain antibody

[0161] This invention provides two single-domain antibodies against IL-23. Through extensive screening, this invention provides two specific single-domain antibodies against IL-23 with high binding affinity. Furthermore, through humanization, corresponding humanized single-domain antibodies against IL-23 are provided.

[0162] The camel-derived single-domain antibody obtained by screening in this invention is named Nb10-9. Based on Nb10-9, humanization was performed to obtain modified humanized single-domain antibodies, named HuNb10-9-1, HuNb10-9-2, and HuNb10-9-3, respectively. The VHH chain of Nb10-9 and its modified humanized single-domain antibodies contains CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:4. Specifically, the VHH chain of Nb10-9 has the amino acid sequence shown in SEQ ID NO:1, while HuNb10-9-1, HuNb10-9-2, and HuNb10-9-3 have the amino acid sequences shown in SEQ ID NO:9-11, respectively. Furthermore, Nb10-9 and its humanized versions all exhibit blocking activity against the binding of IL-23 and IL-23R.

[0163] The camel-derived single-domain antibody obtained by screening in this invention is named Nb11-31. Based on Nb11-31, humanization was performed to obtain a modified humanized single-domain antibody, named HuNb11-31. The VHH chain of Nb10-9 and its modified humanized single-domain antibody contains CDR1 shown in SEQ ID NO:20, CDR2 shown in SEQ ID NO:21, and CDR3 shown in SEQ ID NO:22. The VHH chain of Nb11-31 has the amino acid sequence shown in SEQ ID NO:19, and HuNb11-31 has the amino acid sequence shown in SEQ ID NO:26.

[0164] This invention also provides a heterobivalent comprising two VHH chains derived from the aforementioned two anti-IL-23 single-domain antibodies. In one embodiment of the invention, the heterobivalent is formed by linking the VHH chains of HuNb10-9-1 and HuNb11-31 together via a linker peptide (GGGGS)3, and its amino acid sequence is shown in SEQ ID NO:28. In another embodiment of the invention, the heterobivalent is formed by linking the VHH chains of HuNb10-9-2 and HuNb11-31 together via a linker peptide (GGGGS)3, and its amino acid sequence is shown in SEQ ID NO:29. In yet another embodiment of the invention, the heterobivalent is formed by linking the VHH chains of HuNb10-9-3 and HuNb11-31 together via a linker peptide (GGGGS)3, and its amino acid sequence is shown in SEQ ID NO:30. The present invention unexpectedly discovered that heterobivalents obtained by tandemly combining the VHHs of two single-domain antibodies have significantly superior functional activities compared to commercially available IL-23p19 monoclonal antibodies (e.g., Risankizumab and Guselkumab), including blocking activity against the binding of IL-23 to IL-23R and activity against IL-23-stimulated production of IL-17.

[0165] Furthermore, the aforementioned heterobivalent antibody was fused together with the anti-TL1A single-domain antibody and the Fc of the antibody to construct a bispecific antibody against IL-23 and TL1A. Specifically, the anti-TL1A single-domain antibody used in this invention has the amino acid sequence shown in SEQ ID NO:35. The constructed bispecific antibody exhibits excellent IL-23 binding and blocking activity, and can be applied to the treatment of IL-23 and TL1A, playing a synergistic role in disease treatment.

[0166] Pharmaceutical Composition

[0167] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-described antibody or its active fragment, or its fusion protein, or its antibody-drug conjugate, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0168] The pharmaceutical compositions of the present invention can be directly used to bind to TL1A protein molecules, blocking the TL1A / DR3 pathway, and therefore can be used to treat IL-23-related diseases, such as inflammatory and immune diseases. Furthermore, other therapeutic agents can be used concurrently.

[0169] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0170] In one embodiment of the invention, when using the pharmaceutical composition, a safe and effective amount of the antibody or antibody-drug conjugate of the invention is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0171] Reagent test kit

[0172] The present invention also provides a kit containing the antibody (or fragment thereof) or detection reagent of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0173] This invention also provides a detection kit for detecting IL-23 levels. The kit includes an antibody that recognizes the IL-23 protein (the antibody described in this invention), a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0174] Detection methods

[0175] The present invention also relates to a method for detecting IL-23 protein. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of IL-23 protein in the dissolved samples.

[0176] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0177] application

[0178] As described above, the single-domain antibody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of IL-23-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of IL-23. For example, it can be used for IL-23-mediated immune and inflammatory diseases, including but not limited to: inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; prevention and / or treatment of psoriasis, rheumatoid arthritis, and multiple sclerosis.

[0179] The main advantages of this invention include:

[0180] (1) The IL-23p19 single-domain antibody described in this invention has better IL-23 / IL-23R pathway blocking activity compared with existing reported monoclonal antibodies, and has significant reporter gene cell inhibitory activity and inhibitory activity against IL-17 release from mouse spleen cells.

[0181] (2) The IL23p19 single-domain antibody with excellent activity described above is composed of two IL23p19 single-domain antibodies with different epitopes in series, one of which is a blocking antibody and the other is a non-blocking antibody.

[0182] (3) The IL-23p19 single-domain antibody described in this invention has good binding activity with a variety of IL-23p19 proteins, including IL-23p19 derived from humans and cynomolgus monkeys.

[0183] Example 1: Preparation of IL-23p19 single-domain antibody

[0184] The IL-23 protein sequence was obtained from the Uniprot website (codes P29460 and AAG37232). The 23-328 amino acid sequence of the human IL-23p40 subunit (code P29460) was fused with the 20-189 amino acid sequence of the human IL-23p19 subunit (code AAG37232). The two sequences were linked by a 21-amino acid linker, and a 6xHis tag was added to the C-terminus. This gene was cloned into the pCDNA3.1 vector and transfected into Expi293 cells for expression. After 5 days of transient culture, the supernatant of Expi293 cells was incubated with a nickel column, and the eluted target protein was collected and its purity was determined by SEC-HPLC. The obtained high-purity antigen was mixed with an adjuvant and injected into three healthy Bactrian camels from Xinjiang. Immunization was performed weekly for a total of 7 weeks, followed by a 100 mL peripheral blood sample from each camel. Peripheral blood PBMCs were extracted and total RNA was isolated. After reverse transcription into cDNA, the VHH gene fragment was amplified by two rounds of PCR, cloned into the pMECS vector, and then electroporated into E. coli TG1 competent cells to generate a phage display IL-23 single-domain antibody library.

[0185] Linearized human IL-23 protein and native human IL-23 protein were coated onto ELISA plates and incubated with phage libraries at room temperature for 1 hour, followed by 20 PBST washes to remove non-specific phages. After washing, the eluted phages were used to elute the specifically bound antibodies and display the phages. The eluted phages were then used to infect E. coli TG1 cells in the logarithmic growth phase to expand the phage culture for the next round of screening. The first two rounds of screening used native human IL-23 protein, and the third round used cynomolgus monkey IL-23 protein. After three rounds of screening, significant enrichment of specific phages was obtained. Subsequently, 1800 clones were randomly selected from the phage pools of the second and third rounds for blocking ELISA identification. Positive clones with IL-23 and IL-23R binding blocking activity were sequenced, ultimately yielding single-domain antibodies from six different families.

[0186] Example 2: Species Cross-Reactivity Identification of IL-23p19 Single-Domain Antibodies

[0187] Six candidate single-domain antibodies were expressed in E. coli and their species activity was identified. Human and cynomolgus monkey IL-23 protein were coated onto ELISA plates at 2 μg / mL and incubated overnight at 4°C, followed by blocking with 1% BSA at room temperature for 2 h. The six serially diluted antibodies were added to the blocked ELISA plates and incubated at 37°C for 1 h. The plates were washed three times with PSBT, and then diluted mouse anti-HA antibody was added and incubated at 37°C for 30 min. After washing three times with PSBT, anti-mouse-HRP antibody was added and incubated at 37°C for 30 min. After washing three times with PSBT, TMB was added for color development, and the reaction was terminated with 2M sulfuric acid. The absorbance at OD450 nm was measured using an ELISA reader.

[0188] The results showed that among the six candidate IL-23p19 single-domain antibodies, four (Nb4-43, Nb10-9, Nb11-31, and Nb17-64) exhibited good binding activity with both human and cynomolgus monkey IL-23p19. Figure 1 and Figure 2 ).

[0189] Example 3: Identification of the blocking activity of IL-23p19 single-domain antibody

[0190] The four candidate IL23p19 single-domain antibodies from Example 2 were used to identify the reporter gene cell functional activity. A mixture of candidate IL23p19 single-domain antibodies and human IL-23 protein was prepared using 1640 medium: the serially diluted single-domain antibodies were mixed with a fixed concentration of human IL-23 protein and incubated for 30 min at room temperature; then, the mixture was added to the HEK-293 / IL-23 reporter gene cell line (which expresses IL-23R on its surface), with 40 μL of cell suspension added to each well of a 96-well plate at a cell density of 1.25 E6 / mL; after incubating the 96-well plate at 37°C for 6 h, 80 μL of fluorescence detection reagent was added to each well, and after lysis for 5 min, 100 μL of supernatant was collected from each well and added to a 96-well plate for absorbance measurement using a microplate reader.

[0191] The results are as follows Figure 3As shown: The candidate IL-23p19 single-domain antibody Nb10-9 has good IL-23 / IL-23R blocking activity, and its activity is superior to the reported control IL-23p19 single-domain antibody V900 (see Roberts KJ, Cubitt MF, Carlton TM, et al. Preclinical development of a bispecific TNFα / IL-23neutralising domain antibody as a novel oral treatment for inflammatorybowel disease. Sci Rep. 2021;11(1):19422.Published 2021Sep 30.doi:10.1038 / s41598-021-97236-0).

[0192] Example 4: Antibody Humanization and Heterobivalent Construction

[0193] 4.1 Antibody humanization

[0194] The candidate IL-23p19 single-domain antibody was humanized. The CDR region was labeled using the IMGT annotation method, and the CDR remained unchanged. The backbone region was then humanized, and various reverse mutation schemes were designed. See Table 1 below.

[0195] Table 1. Humanization of candidate IL-23p19 single-domain antibodies

[0196]

[0197] 4.2 Construction of Heterovalent Bodies

[0198] The aforementioned humanized IL-23p19 single-domain antibody was tandemly combined to form a dual-epitope heterobivalent, as shown in Table 2 below. Further, the heterobivalent sequence was fused with Fc and simultaneously combined with a TL1A single-domain antibody (SEQ ID NO: 35) to form a bispecific antibody (knobinto hole), which was then transfected into CHO-S cells for expression and purification.

[0199] Table 2. Combinations of Heterogeneous Divalents

[0200] Bispecific antibody number Humanized IL-23p19 single-domain antibody tandem combination P00703 HuNb10-9-1+HuNb11-31 P00704 HuNb10-9-2+HuNb11-31 P00705 HuNb10-9-3+HuNb11-31 P00706 HuNb10-9-1+HuNb17-64 P00707 HuNb10-9-2+HuNb17-64 P00708 HuNb10-9-3+HuNb17-64 P00709 HuNb10-9-1+HuNb4-43-1 P00710 HuNb10-9-2+HuNb4-43-1 P00711 HuNb10-9-3+HuNb4-43-1 P00712 HuNb10-9-1+HuNb4-43-2 P00713 HuNb10-9-2+HuNb4-43-2 P00714 HuNb10-9-3+HuNb4-43-2 P00715 HuNb10-9-1+HuNb4-43-3 P00716 HuNb10-9-2+HuNb4-43-3 P00717 HuNb10-9-3+HuNb4-43-3

[0201] Example 5: Identification of the functional activity of IL-23p19 antibody using the reporter assay

[0202] The bispecific antibody expressed and purified in Example 4 was used to identify the functional activity of IL-23p19. A mixture of candidate antibody and human IL-23 protein was prepared using 1640 medium: the serially diluted bispecific antibody was mixed with 1 ng / mL human IL-23 protein and incubated for 30 min at room temperature; then, it was added to the HEK-293 / IL-23 reporter gene cell line, with 40 μL of cell suspension added to each well of a 96-well plate at a cell density of 1.25 E6 / mL; after incubating the 96-well plate at 37°C for 6 h, 80 μL of fluorescent detection reagent was added to each well, and after lysis for 5 min, 100 μL of supernatant was collected from each well and added to a 96-well plate for absorbance measurement using a microplate reader.

[0203] Table 3. Results of HEK-293 / IL-23 reporter gene cell line detection

[0204]

[0205]

[0206] Table 4. Results of HEK-293 / IL-23 reporter gene cell line detection

[0207] <![CDATA[IC 50 / nM]]> P00703 0.08020 P00707 0.03171 P00714 2.870 Risankizumab 0.01262

[0208] The results are as follows Figures 4A-4C As shown in Tables 3 and 4, candidate antibodies P00704 and P00705 exhibit significantly superior blocking activity compared to marketed IL-23p19 monoclonal antibodies Risankizumab and Guselkumab.

[0209] The activity of the candidate antibodies in the signaling pathway induced by cynomolgus monkey IL-23 was further identified. A mixture of candidate antibodies and cynomolgus monkey IL-23 protein was prepared using 1640 medium: The serially diluted bispecific antibody was mixed with 1 ng / mL cynomolgus monkey IL-23 protein and incubated for 30 min at room temperature; then, it was added to the HEK-293 / IL-23 reporter gene cell line, with 40 μL of cell suspension added to each well at a cell density of 1.25 E6 / mL into a 96-well plate; after incubating the 96-well plate at 37°C for 6 h, 80 μL of fluorescent detection reagent was added to each well, and after lysis for 5 min, 100 μL of supernatant was collected from each well and added to a 96-well plate for absorbance measurement using a microplate reader.

[0210] The results are as follows Figure 5 As shown, candidate antibodies P00704 and P00705 still exhibit superior blocking activity compared to marketed IL-23p19 monoclonal antibodies Risankizumab and Guselkumab.

[0211] Example 6: Specificity Identification of Candidate Antibodies

[0212] Biotinylated P00704, P00705, Ustekinumab, and Risankizumab antibodies were prepared to a final concentration of 2 μg / mL using 0.05% PBST as loading solutions. Two different IL-12s (native IL-12 and linearized single-chain scIL-12) were diluted to 2 μg / mL using 0.05% PBST as association solutions. A blank of 0.05% PBST served as a baseline. All materials were placed in 96-well plates. The SA probe was pre-wetted in 0.05% PBST for 10 min; then, the plates were sequentially run in the baseline for 180 s, in the loading solution for 220 s, in the baseline solution for 180 s, and in the association solution for 240 s. Binding detection was performed on a Fortebio instrument.

[0213] The results are as follows Figure 6 and Figure 7 As shown, candidate antibodies P00704 and P00705 do not bind to IL12, thus exhibiting good specificity.

[0214] Example 7: Inhibitory activity of humanized antibody against mouse spleen mIL17

[0215] Spleens from C57BL / 6 mice were homogenized in pre-chilled RPMI-1640 medium and filtered through a 40µm cell mesh. The homogenate was centrifuged at low temperature, and the supernatant was discarded. Cells were resuspended in 2mL of ACK erythrocyte lysis buffer and incubated at room temperature for 3 min. Five volumes of ACK lysis buffer were added to RPMI-1640 medium and mixed. The supernatant was discarded, and cells were resuspended in RPMI-1640 complete medium, diluted, and seeded into 96-well plates. 100µL of spleen cells containing 2ng / mL human IL23, 100ng / mL IL-2, and 2ng / mL IL-23p19 antibody (serialized 4-fold starting from 2000pM) were added and mixed. The cells were incubated at 37°C with 5% CO2 for 72 h. After centrifugation, the supernatant was transferred to a new 96-well plate, and the mIL-17 content in the supernatant was measured according to the method described in the BD mIL-17 assay kit.

[0216] The results are as follows Figure 8 As shown, P00704 and P00705 both have significant inhibitory effects on IL-17 produced by IL-23 stimulation in mice, and their activity is superior to that of the control antibodies Risankizumab and Guselkumab.

[0217] Antibody sequence information of the present invention

[0218]

[0219]

[0220]

[0221]

[0222] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-IL-23 single-domain antibody or its antigen-binding fragment, characterized in that, The VHH chain of the anti-IL-23 single-domain antibody or its antigen-binding fragment contains a complementarity-determining region (CDR) selected from the group consisting of: (Z1) CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4; or (Z2) CDR1 shown in SEQ ID NO:20, CDR2 shown in SEQ ID NO:21 and CDR3 shown in SEQ ID NO:

22.

2. A fusion protein, characterized in that, The structure of the fusion protein from the N-terminus to the C-terminus is shown in formula Ia or Ib: ALB(Ia); BLA(Ib); in, A is the anti-IL-23 single-domain antibody or its antigen-binding fragment as described in claim 1; B is the Fc fragment of IgG; and L represents no or flexible joints.

3. A multispecific antibody, characterized in that, The multispecific antibody comprises the anti-IL-23 single-domain antibody as described in claim 1 or its antigen-binding fragment, or the fusion protein as described in claim 2.

4. The multispecific antibody as described in claim 3, characterized in that, The multispecific antibody is a bispecific antibody, which includes the anti-IL-23 single-domain antibody or its antigen-binding fragment as described in claim 1, and the anti-TNF-like ligand 1A (TL1A) single-domain antibody.

5. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the anti-IL-23 single-domain antibody or its antigen-binding fragment as described in claim 1; and (ii) Optional tag sequences to assist in expression and / or purification.

6. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: the anti-IL-23 single-domain antibody as described in claim 1 or its antigen-binding fragment, the fusion protein as described in claim 2, the multispecific antibody as described in claim 3 or 4, or the recombinant protein as described in claim 5.

7. An expression vector comprising the polynucleotide as described in claim 6.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the anti-IL-23 single-domain antibody as claimed in claim 1 or its antigen-binding fragment, the fusion protein as claimed in claim 2, the multispecific antibody as claimed in claim 3 or 4, or the recombinant protein as claimed in claim 5; and (ii) Pharmaceutically acceptable carriers.

9. An IL-23 inhibitor, characterized in that, The inhibitor comprises the anti-IL-23 single-domain antibody as described in claim 1 or its antigen-binding fragment, the fusion protein as described in claim 2, the multispecific antibody as described in claim 3 or 4, or the recombinant protein as described in claim 5.

10. Use of the anti-IL-23 single-domain antibody of claim 1 or its antigen-binding fragment, the fusion protein of claim 2, the multispecific antibody of claim 3 or 4, or the recombinant protein of claim 5, for the preparation of: (a) Medications used to prevent and / or treat IL-23-mediated diseases or conditions; or (b) Detection reagents, test plates or test kits for detecting IL-23 molecules.

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