Use of Anti-ox40 antibody in treatment of dermatitis

Abstract

The present application relates to a use of an anti-OX40 antibody or an antigen-binding fragment thereof in the treatment of dermatitis. The anti-OX40 antibody or the antigen-binding fragment thereof comprises heavy chain variable regions CDR1, CDR2, and CDR3 and light chain variable regions CDR1, CDR2, and CDR3.

Description

Use of anti-OX40 antibodies in the treatment of dermatitis Technical Field

[0001] This application relates to the field of biomedicine, specifically to the use of an anti-OX40 antibody in the treatment of dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is the most common chronic inflammatory skin disease, affecting both adults and children, with a global prevalence of up to 20% (NPL 1). Standard care for skin inflammation includes topical treatments such as topical corticosteroids or tacrolimus ointment. Oral immunosuppressants (such as cyclosporine) and glucocorticoids (such as systemic corticosteroids) have also proven effective in treating refractory AD cases, but are sometimes accompanied by serious toxicity and side effects; therefore, this regimen is currently strictly limited to short-term and / or intermittent treatment.

[0003] OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor gene family. OX40 is predominantly expressed early after antigen activation of T cells, including CD4 and CD8 positive T cells; type 1, 2, and 17 helper T cells; and forkhead box P3 (FoxP3) positive / CD4 positive regulatory T cells. OX40 is involved in the expansion and survival of antigen-specific T cells. The ligand of OX40 (OX40L) is primarily expressed on activated antigen-presenting cells and endothelial cells during inflammation. In allergic dermatitis, including atopic dermatitis, T cells, basophils, or mast cells are often in a hyperactivated state due to environmental factors, leading to the excessive secretion of inflammatory cytokines (such as IL-4, IL-13, and IL-5) and other chemical mediators (such as histamine and serotonin), thereby triggering skin inflammation. Studies have found that OX40 is a type of membrane protein that is highly expressed after T cell activation. OX40 can be linked to OX40L to lead to enhanced T cell survival and proliferation. Therefore, regulating T cell subsets, such as Th2, through OX40 (CD134) may play a role in the pathology of allergic inflammatory skin diseases, such as atopic dermatitis.

[0004] Therefore, there is an urgent need to obtain a safe and effective OX40-targeted drug for the treatment of dermatitis, especially atopic dermatitis. Summary of the Invention

[0005] This application provides the use of an anti-OX40 antibody or its antigen-binding fragment, or a nucleic acid encoding the antibody, with good specificity, high affinity, and stability in the treatment of dermatitis diseases such as allergic dermatitis and atopic dermatitis.

[0006] The anti-OX40 antibody described in this application can block the overactivation of dermatitis-associated immune cells, such as Th2 cells, thereby exerting a therapeutic function in inflammatory skin diseases, such as allergic dermatitis and atopic dermatitis.

[0007] The anti-OX40 antibody or its antigen-binding fragment described in this application has one or more of the following advantages when treating patients with allergic dermatitis, especially atopic dermatitis: significant relief of dermatitis symptoms, low toxicity, significant reduction of inflammatory cytokine levels, and durable therapeutic effect, and has a very broad application prospect.

[0008] On one hand, this application provides the use of an anti-OX40 antibody or its antigen-binding fragment in the preparation of a medicament for treating dermatitis, wherein the anti-OX40 antibody or its antigen-binding fragment comprises a CDR region selected from any of the following groups:

[0009] 1) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 5-7, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 14-16, respectively.

[0010] 2) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 23-25, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 32-34, respectively.

[0011] 3) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 41-43, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 50-52, respectively.

[0012] 4) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 63, 42, and 43, respectively, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 50-52, respectively.

[0013] 5) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 41, 67, and 43, respectively, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 50-52, respectively.

[0014] 6) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 41, 42, and 70, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 50-52, respectively.

[0015] 7) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 63, 67, and 70, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 75-77, respectively.

[0016] 8) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 41, 42, and 70, respectively, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 75-77, respectively.

[0017] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment comprises a heavy chain variable region of the following group, the sequences of which are shown in SEQ ID NO: 4, 22, 40, 62, 66, 72 and 79.

[0018] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment comprises a group of light chain variable regions, the sequences of which are shown in SEQ ID NO: 13, 31, 49, 74 and 81.

[0019] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 79 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 81.

[0020] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment is a whole antibody, a bispecific antibody, scFv, Fab, Fab', F(ab')2, or Fv.

[0021] In some embodiments, when the anti-OX40 antibody or its antigen-binding fragment is scFv, a linker peptide may be present between the variable regions of its heavy and light chains.

[0022] In some embodiments, the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment is selected from IgG, IgM, IgE, IgD, and IgA.

[0023] In some embodiments, the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment is selected from IgG1 or IgG4.

[0024] In some embodiments, the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment is selected from IgG1, and the heavy chain constant region of IgG1 contains mutations of S239D, I332E and / or A330L.

[0025] In some embodiments, the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment contains an amino acid sequence as shown in any one of SEQ ID NO: 82-84.

[0026] In some embodiments, the light chain constant region of the anti-OX40 antibody or its antigen-binding fragment is κ or λ.

[0027] In some embodiments, the light chain constant region of the anti-OX40 antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO: 85.

[0028] In some embodiments, the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO: 83, and the light chain constant region contains an amino acid sequence as shown in SEQ ID NO: 85.

[0029] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment is deglycosylated.

[0030] In some embodiments, the type of glycosylation modification includes N-position glycosylation or O-position glycosylation.

[0031] In some embodiments, the deglycosylation modification includes defucosylation modification.

[0032] In some embodiments, the deglycosylation modification site is located at the Fc terminus of the antibody.

[0033] In some embodiments, the defucosylation modification site includes Asn297.

[0034] In some embodiments, the drug comprises a nucleic acid molecule encoding an anti-OX40 antibody or an antigen-binding fragment thereof as described in any of the preceding claims.

[0035] In some embodiments, the drug includes a carrier containing the nucleic acid molecule.

[0036] In some embodiments, the drug comprises a cell containing the nucleic acid molecule or the carrier.

[0037] In some embodiments, the drug comprises a conjugate containing the anti-OX40 antibody or an antigen-binding fragment thereof.

[0038] In some embodiments, the drug comprises a pharmaceutical composition comprising an anti-OX40 antibody or an antigen-binding fragment thereof as described in any of the preceding claims, a nucleic acid molecule as described in the preceding claims, a carrier as described in the preceding claims, a cell as described in the preceding claims, and / or a conjugate as described in the preceding claims, and a pharmaceutically acceptable carrier.

[0039] In some embodiments, the dermatitis includes OX40-mediated dermatitis.

[0040] In some embodiments, the OX40-mediated dermatitis includes allergy-related dermatitis.

[0041] In some embodiments, the allergy-related dermatitis described herein includes, but is not limited to, one or more of the following: urticaria, atopic dermatitis, atopic eczema, contact dermatitis, photodermatitis, white atrophy, allergic purpura, and allergic vasculitis.

[0042] In some embodiments, the dermatitis described therein is atopic dermatitis.

[0043] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0044] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0045] Figures 1A-1D show the induction and treatment of dorsal ear dermatitis in each group of cynomolgus monkeys described in this application. Figure 1A corresponds to the dorsal induction control group, Figure 1B corresponds to the dorsal induction DF004 treatment group, Figure 1C corresponds to the ear induction control group, and Figure 1D corresponds to the ear induction DF004 treatment group.

[0046] Figure 2 shows the skin scores of the cynomolgus monkeys in the model group and the DF004 treatment group described in this application;

[0047] Figure 3 shows the skin scoring criteria for cynomolgus monkey dermatitis in each group described in this application;

[0048] Figure 4 shows the skin pathology scores of the cynomolgus monkeys in the model group and the DF004 treatment group described in this application;

[0049] Figure 5 shows the curves of weight change ratio of cynomolgus monkeys in the model group and the DF004 treatment group described in this application.

[0050] Figure 6 shows the ear thickness variation curves of the cynomolgus monkeys in the model group and the DF004 treatment group described in this application.

[0051] Figure 7 shows the levels of inflammatory cytokines in the skin of cynomolgus monkeys in the model group and the DF004 treatment group described in this application;

[0052] Figure 8 shows the skin scores of the cynomolgus monkeys in the model group, DF004 treatment group, and DF004-FKO treatment group described in this application. Detailed Implementation

[0053] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0054] Terminology Definition

[0055] In this application, the term "OX40" generally refers to a tumor necrosis factor family protein, also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4), ACT35, IMD16, TXGP1L, and CD134. OX40 refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise stated. OX40 is a type I transmembrane glycoprotein primarily expressed on activated CD4+ T cells and CD8+ T cells. For example, the Uniprot accession number for human OX40 could be P43489.

[0056] In this application, the term "OX40L" generally refers to the ligand of OX40, also known as the ACT-4 receptor, CD134L, CD252, and Gp34. OX40L is a type II transmembrane glycoprotein expressed on activated antigen-presenting cells, such as dendritic cells and B cells. For example, the Uniprot accession number for human OX40L is P23510. OX40 / OX40L signaling plays a crucial role in the activation, proliferation, and inhibition of apoptosis of T cells.

[0057] In this application, the terms "specific binding" or "specific" generally refer to measurable and reproducible interactions, such as binding between a target and an antibody, where the presence of the target is determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to the target with greater affinity, strength, ease, and / or duration than it binds to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins of different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding.

[0058] In this application, the term "CDR," also known as "complementarity-determining region," typically refers to a region within the variable structural domain of an antibody whose sequence is highly variable and / or forms a structurally defining loop. Typically, antibodies comprise six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel antibodies consisting only of the heavy chain can function normally and stably even in the absence of the light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art. For example, the amino acid sequence numbering of the antigen-binding protein can be in accordance with the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310; Lefranc et al., 2005, DevComp Immunol 29:185-203). For example, the CDR of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). The boundaries of the CDR of the variable region of the same antibody obtained based on different coding systems may differ. That is, the CDR sequence of the variable region of the same antibody defined under different coding systems is different.Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined in this invention due to the application of different schemes (e.g., different coding systems). The CDR boundaries of the antibodies of this invention can be determined manually by any scheme or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0059] In this application, the terms "variable domain" and "variable region" are used interchangeably, generally referring to a portion of the antibody heavy chain and / or light chain. The variable domains of the heavy and light chains can be respectively referred to as "V..." H " and "V L (or referred to as "VH" and "VL" respectively). These domains are usually the most varied parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0060] In this application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ significantly in sequence between antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire variable domain. It is typically concentrated in three segments within the light and heavy chain variable domains, known as hypervariable regions (CDRs or HVRs). The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, most of which are β-sheet conformations linked by three CDRs forming a ring link, and in some cases forming part of a β-sheet structure. The CDRs in each chain are held together closely by the FR regions, and CDRs from the other chain together promote the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0061] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof or a derivative thereof, encompassing any polypeptide including an antigen-binding site, whether it is produced in vitro or in vivo. This term includes, but is not limited to, polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "complete," such as in "complete antibody," for the purposes of this invention, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that maintain antigen-binding function (e.g., specific binding to OX40). Typically, such fragments should include an antigen-binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units and a polypeptide chain called the J chain, containing 10 antigen-binding sites. IgA antibodies consist of 2-5 basic tetrameric units that can polymerize with the J chain to form multivalent combinations. For IgG, a tetrameric unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds depending on the H chain isoform. Each H and L chain also has regularly spaced intrachain disulfide bridging bonds. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for α and γ chains, and four CH domains for μ and ε isoforms. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains. VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. L chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domain. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of their heavy chain (CH) constant domain. Currently, five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM, with heavy chains named α, δ, ε, γ, and μ, respectively.

[0062] In this application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind an antigen (e.g., OX40). In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb.

[0063] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, an intact antibody can be digested using papain. After papain digestion, the antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as above). The Fab fragment can consist of a complete L chain with a variable region of a heavy chain and the H chain (V... H The first constant region (C) H 1) Composition.

[0064] In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is slightly larger than the Fab fragment. For example, the Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, the Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0065] In this application, the term "F(ab')2" generally refers to an antibody fragment produced by digesting an intact antibody with pepsin. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. The F(ab')2 fragment has bivalent antigen-binding activity and is capable of cross-linking antigens.

[0066] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy chain variable region and light chain variable region, and lacking the heavy chain constant region and light chain constant region. The heavy chain variable region and light chain variable region include, for example, CDRs. For example, an Fv fragment comprises all or part of the amino-terminal variable region of about 110 amino acids of the heavy and light chains.

[0067] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment including a variable region comprising a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used in this application, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0068] In this application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having either a VH domain or a VL domain, as seen in, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and other published patent applications, such as WO 06 / 030220, WO 06 / 003388, and Domantis Ltd. The term "dAb" generally includes sdAb. The term "sdAb" generally refers to a single-domain antibody. A single-domain antibody generally refers to an antibody fragment consisting only of the variable region (VH domain) of the antibody heavy chain or the variable region (VL) of the antibody light chain.

[0069] In this application, the term "VHH" generally refers to the variable antigen-binding domain of heavy chain antibodies derived from camels (camels, dromedaries, llamas, alpacas, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302; and review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH may also be referred to as nanobody (Nb).

[0070] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation consisting of a single molecule. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of antibodies through any particular method. For example, the monoclonal antibodies used in this application can be prepared in hybridoma cells or through recombinant DNA methods.

[0071] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody from a laboratory animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in human individuals compared to parental (e.g., alpaca-derived) antibodies.

[0072] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., an alpaca antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids within the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of the constant region of human immunoglobulins. "Humanized antibodies" retain antigen specificity similar to the original antibody. The "humanized" form of a non-human (e.g., alpaca) antibody may minimally contain a chimeric antibody with a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues from a non-human species (donor antibody) (such as an alpaca, mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications not found in receptor antibodies or in donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0073] In this application, the term "fully human antibody" generally refers to an antibody that contains only the protein sequence of human immunoglobulins. If it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells, then a fully human antibody may contain mouse glycans. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be generated in humans or in transgenic animals with human immunoglobulin germline sequences by phage display or other molecular biology methods. Exemplary techniques that can be used to manufacture antibodies are described in U.S. Patents: 6,150,584, 6,458,592, and 6,420,140. Other techniques, such as the use of libraries, are known in the art.

[0074] In this application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue isolated from its natural environment or synthesized artificially.

[0075] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle that can insert a polynucleotide encoding a protein into itself and enable the protein to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material elements they carry to be expressed within the host cells. For example, vectors can include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors can include retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. The carrier may also include components that help it enter the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0076] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this invention. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). Cells may include cells transfected in vitro using the vectors described in this application. Cells may be bacterial cells (e.g., *E. coli*), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, and NSO cells.

[0077] In this application, the term "conjugate" generally refers to a conjugate formed by combining the other reagent (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents) with the antibody or its antigen-binding fragment (e.g., covalently linked by a linker molecule), which can deliver the other reagent to the target cell (e.g., tumor cells) by specifically binding the antibody or its antigen-binding fragment to the antigen on the target cell.

[0078] In this application, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the antibody or antigen-binding fragment described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0079] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions such as sodium; and / or nonionic surfactants.

[0080] In this application, the term "allergic dermatitis" refers to a chronic inflammatory skin disease associated with elevated levels of immunoglobulin E (IgE) in the patient's body. Allergic dermatitis is usually caused by external allergens inducing abnormal secretion of pro-inflammatory factors by cells in skin tissue. These pro-inflammatory factors can be IL-, such as IL-4, IL-5, IL-13, etc., or vasoactive amines (such as histamine), enzymes, lipid molecules (such as PGD2, LTs), etc. Typically, allergic dermatitis is induced by the following: in the presence of allergens, the patient's type 2 helper T cells are overactivated, inducing a type 2 helper T cell-mediated adaptive immune response, secreting various type 2 cytokines, such as IL-4, IL-5, and IL-13. These cytokines further promote the maturation and differentiation of B cells, causing effector B cells (plasma cells) to secrete specific IgE antibodies. IgE binds to IgE receptors on mast cells and basophils, secreting vasoactive amines (such as histamine), enzymes, lipid molecules (such as PGD2 and LTs), and more cytokines such as IL-5 and IL-13 to participate in the inflammatory response, thereby causing inflammation in the skin tissue.

[0081] In this application, the term "allergen" refers to the antigenic portion of an antigen or molecule that can trigger an allergic reaction upon contact with an individual. Generally, whether an individual is allergic to an allergen can be determined through clinical trials, estimation based on the subject's medical history, or by any other suitable method known in the art and described in the following examples. An antigen can be called an allergen if a small percentage of individuals exhibit an immune response upon exposure to the molecule. A variety of isolated allergens are known in the art. For example, common allergens in patients with seasonal allergic rhinitis include pollen from grasses, trees, weeds, and mold spores. Common allergens in patients with perennial allergic rhinitis include house dust mites, sawdust, mold, fungal spores, feather pillows, animal dander, animal hair, and most commonly, cigarette smoke.

[0082] In this application, the term "atopic dermatitis (AD)" refers to a chronic, relapsing, pruritic inflammatory skin disease associated with allergens, often accompanied by skin inflammation and skin barrier defects. The outer layer of skin, or stratum corneum, normally functions as a barrier, preventing foreign substances (such as bacteria, viruses, or allergens) from entering the body. However, in AD, this barrier function is weakened, making the body's immune cells more susceptible to inflammatory responses from external foreign substances (such as allergens). Symptoms of AD include itching, erythema, edema, dryness, erosion / epidermal shedding, purulent exudation and crusting, lichenification, impaired skin barrier, and redness. AD can be classified as mild, moderate, or severe based on skin coverage and symptom severity. Mild AD affects a small area of ​​skin and may occasionally cause itching or redness. However, moderate and severe AD cover a larger area of ​​skin and involve more frequent itching, with periods of intense itching. For an overview of disease severity and quality of life indicators for Alzheimer's disease (AD), see, for example, Rehal and Armstrong (2011), Plos ONE 6(4): e17520 and Gooderham et al. (2018), and J Cutan Med Surg., 22(IS) 10S-16S). Many diagnostic criteria for AD exist internationally: Hanifin & Rajka criteria, Kangtian criteria, Schultz-Larsen criteria, Lillehammer criteria, Williams criteria, the International Study of Childhood Asthma and Allergy (ISAAC) questionnaire, Japanese Dermatological Society criteria, Diepgen criteria, Millennium criteria, Danish Allergy Research Centre (DARC) criteria, and Chinese criteria.

[0083] In this application, the terms "patient," "subject," "individual," etc., are used interchangeably herein and include humans and other animals. For example, they include mammals. For example, they include humans.

[0084] In this application, the term "prevention and / or treatment" includes not only the prevention and / or treatment of a disease, but also generally includes preventing the onset of the disease, slowing or reversing the disease progression, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated with it, and / or preventing further increase in the severity of the disease and / or any symptoms associated with it, and preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the patient being treated.

[0085] In this application, the term "relief" generally refers to a reduction, shortening, or delay of a symptom, disease, condition, or phenotype. The symptom, disease, condition, or phenotype may include subjective perceptions of the subject, such as pain, dizziness, or other physiological disturbances, or medically detectable indicators, such as lesions detected by medical testing, for example, abnormal B cell and / or plasma cell activity, autoantibody expression levels, platelet count, or bleeding.

[0086] In this application, the terms "patient," "subject," and "organism" are used interchangeably, including human subjects and other mammalian subjects receiving preventive or therapeutic treatment. In this application, the term "patient suffering from..." generally refers to a patient exhibiting clinical signs of a disease.

[0087] In this application, the terms "application" and "administration" are used interchangeably and generally refer to the act of applying a substance to a subject by methods described herein or otherwise known in the art. Any method known to those skilled in the art for contacting cells, organs, or tissues with the substance may be employed. For example, dosage forms include injections, tablets, capsules, oral liquids, granules, ointments, suppositories, or aerosols. For example, methods of administration include aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. For example, administration includes intravenous injection, intraperitoneal injection, subcutaneous injection, or intramuscular injection.

[0088] In this application, the proteins, peptides and / or amino acid sequences involved should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or peptide.

[0089] In this application, the terms "effective amount" or "effective dose" generally refer to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective amount" or "therapeutic effective dose" of a drug or therapeutic agent generally refers to any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease remission (proven by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of damage or disability due to the disease). A "preventive effective amount" or "preventive effective dose" of a drug generally refers to an amount of drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of disease progression or relapse, inhibits the development or relapse of the disease. The ability of a therapeutic agent or preventive agent to promote disease remission or inhibit disease progression or relapse can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, predicting efficacy in humans in animal model systems, or by determining the activity of the agent in an in vitro assay.

[0090] In this application, the variant can be, for example, a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to OX40 or a fragment thereof). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conserved substitution.

[0091] In this application, the homolog can be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to OX40 or a fragment thereof).

[0092] In this application, homology generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence homology percentage" can be calculated by comparing two sequences to be aligned within a comparison window, determining the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence homology percentage. Alignments performed to determine the sequence homology percentage can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. Homology can also be determined using FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and DJ Lipman, “An Improved Tool for Biological Sequence Alignment,” Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and DJ Lipman and W.R. Pearson, “A Fast and Sensitive Search for Protein Similarity,” Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, “A Basic Tool for Local Alignment Search,” Journal of Molecular Biology, 215: 403-410, 1990.

[0093] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0094] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0095] In this application, the term "deglycosylation modification" generally refers to the removal or reduction of the content of a protein linked to a glycan. In some embodiments, the protein includes an anti-OX40 antibody or its antigen-binding fragment. The glycan may be composed of a single type of monosaccharide, including galactose, fucose, high-mannose, N-acetylneuraminic acid (NANA), and N-glycosylneuraminic acid, etc. The glycan may also be a complex oligosaccharide composed of multiple monosaccharides. The glycosylation type includes N-position glycosylation and O-position glycosylation. The deglycosylation modification site may be located at the Fc terminus of the antibody, for example, at the Asn297 position of the antibody. Defucosylation modification generally refers to the removal or reduction of the content of the antibody or its antigen-binding fragment bound to fucose, for example, a reduction of 10%, 20%, 30%, 50%, or more.

[0096] Invention Details

[0097] This application provides the use of an anti-OX40 antibody or its antigen-binding fragment, or a nucleic acid encoding the antibody, with good specificity, high affinity, and stability in the treatment of dermatitis diseases such as allergic dermatitis and atopic dermatitis.

[0098] Current treatments for atopic dermatitis include conventional systemic immunosuppressants such as corticosteroids, cyclosporine, or methotrexate. However, these medications do not target the specific sites of immune dysregulation in atopic dermatitis and can lead to serious adverse events, including liver and kidney dysfunction. With further advancements in targeted therapies, interleukin-4 and β13 antibodies, phosphodiesterase-4 inhibitors, and JAK inhibitors have become key areas of focus. In a comparative study, the interleukin-4 antibody dupilumab and the JAK inhibitor abrocitinib were both associated with reduced signs and symptoms of atopic dermatitis compared to placebo. However, numerous adverse reactions exist; for example, many patients experience long-term functional conjunctivitis after dupilumab treatment, especially when combined with seasonal allergic conjunctivitis. JAK inhibitors carry risks of thromboembolism and cancer and may be associated with respiratory infections, herpes zoster infections, headache, nausea, diarrhea, and decreased white blood cell counts. These new therapies should only be used cautiously after evaluation based on the specific patient's condition, especially in sensitive populations (children) and patients with typical complications of atopic dermatitis.

[0099] This application provides an effective and safe OX40 antibody for treating atopic dermatitis. The anti-OX40 antibody described in this application targets the relevant immune cells upstream of atopic dermatitis and can block the overactivation of dermatitis-related immune cells, such as Th2 cells, thereby exerting a therapeutic function in inflammatory skin diseases such as allergic dermatitis and atopic dermatitis.

[0100] The direct mechanism of atopic dermatitis is inflammation associated with Th2 cell dysregulation. Activated T cells release cytokines into the skin, primarily interleukin-4 and 13, which activate the downstream Janus kinase (JAK) pathway, further activating B cells and plasma cells to promote inflammation, itching, and the production of antigen-specific IgE. Existing treatments often target downstream cytokines and activation pathways, while the anti-OX40 antibody described in this application can block the overactivation of upstream dermatitis-associated immune cells, such as Th2 cells, thereby achieving a therapeutic effect on atopic dermatitis.

[0101] The anti-OX40 antibody or its antigen-binding fragment described in this application has one or more of the following advantages when treating patients with allergic dermatitis, especially atopic dermatitis: significant relief of dermatitis symptoms, low toxicity, significant reduction of inflammatory cytokine levels, and durable therapeutic effect, and has a very broad application prospect.

[0102] Antibody or antigen-binding fragment

[0103] On one hand, this application provides the use of anti-OX40 or its antigen-binding moiety in the preparation of a medicament for treating OX40-mediated diseases. For example, the diseases may include inflammatory skin diseases.

[0104] This invention utilizes yeast display technology to obtain an anti-OX40 antibody with good specificity, high affinity, and stability through screening and further affinity maturation. This antibody can specifically bind to human OX40, and by binding to activated T cells, it can enhance T cell activation, thus significantly inhibiting tumor growth. Furthermore, the anti-OX40 antibody of this invention is a fully human antibody, which, compared with traditional murine, chimeric, and humanized antibodies, has lower immunogenicity, reduces patient rejection, and has better drug-like properties.

[0105] The anti-OX40 antibody or its antigen-binding portion described in this application includes a CDR region selected from the group consisting of:

[0106] 1) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 5-7, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 14-16, or its heavy chain variable regions and light chain variable regions satisfy at least one of the following sequences compared with the aforementioned sequences: a) binding to the same antigenic epitope; b) identity greater than 70%, 80%, 85%, 90%, or 97%;

[0107] 2) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 23-25, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 32-34, or its heavy chain variable regions and light chain variable regions satisfy at least one of the following sequences compared with the aforementioned sequences: a) binding to the same antigenic epitope; b) identity greater than 70%, 80%, 85%, 90%, or 97%;

[0108] 3) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 41-43, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 50-52, or its heavy chain variable regions and light chain variable regions satisfy at least one of the following sequences compared with the aforementioned sequences: a) binding to the same antigenic epitope; b) identity greater than 70%, 80%, 85%, 90%, or 97%;

[0109] 4) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 63, 42, and 43, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 50-52, respectively. Alternatively, its heavy chain variable regions and light chain variable regions, compared with the aforementioned sequences, satisfy at least one of the following two conditions: a) they bind to the same antigenic epitope; b) the identity is greater than 70%, 80%, 85%, 90%, or 97%.

[0110] 5) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 41, 67, and 43, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 50-52, respectively. Alternatively, its heavy chain variable regions and light chain variable regions, compared with the aforementioned sequences, satisfy at least one of the following two conditions: a) they bind to the same antigenic epitope; b) the identity is greater than 70%, 80%, 85%, 90%, or 97%.

[0111] 6) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 41, 42, and 70, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 50-52, respectively. Alternatively, its heavy chain variable regions and light chain variable regions, compared with the aforementioned sequences, satisfy at least one of the following two conditions: a) they bind to the same antigenic epitope; b) the identity is greater than 70%, 80%, 85%, 90%, or 97%.

[0112] 7) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 63, 67, and 70, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 75-77, respectively. Alternatively, its heavy chain variable regions and light chain variable regions, compared with the aforementioned sequences, satisfy at least one of the following two conditions: a) they bind to the same antigenic epitope; b) the identity is greater than 70%, 80%, 85%, 90%, or 97%.

[0113] 8) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 41, 42, and 70, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 75-77, respectively. Alternatively, its heavy chain variable regions and light chain variable regions, compared with the aforementioned sequences, satisfy at least one of the following two conditions: a) they bind to the same antigenic epitope; b) the identity is greater than 70%, 80%, 85%, 90%, or 97%.

[0114] For example, the anti-OX40 antibody or its antigen-binding portion described in this application further includes a heavy chain variable region framework region selected from the following group:

[0115] 1) The sequences of its heavy chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 8-11, respectively;

[0116] 2) The sequences of its heavy chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 26-29, respectively;

[0117] 3) The sequences of its heavy chain variable regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 44-47, respectively.

[0118] For example, the anti-OX40 antibody or its antigen-binding portion described in this application further includes a light chain variable region framework region selected from the following group:

[0119] 1) The sequences of its light chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 17-20, respectively;

[0120] 2) The sequences of its light chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 35-38, respectively;

[0121] 3) The sequences of its light chain variable regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 53-56, respectively.

[0122] For example, the anti-OX40 antibody or its antigen-binding portion described in this application includes a heavy chain variable region selected from the group consisting of sequences shown in SEQ ID NO: 4, 22 and 40, or a heavy chain variable region that satisfies at least one of the following sequences compared to the aforementioned sequences: a) binding to the same antigenic epitope, b) identity greater than 70%, 80%, 85%, 90% or 97%.

[0123] Furthermore, it includes heavy chain variable regions selected from the group shown in SEQ ID NO: 4, 22 and 40, or heavy chain variable regions CDR regions that satisfy at least one of the following two conditions compared to the aforementioned sequences: a) binding to the same antigenic epitope; b) the number of mutated amino acids not exceeding 3.

[0124] Furthermore, it includes heavy chain variable regions selected from the group shown in SEQ ID NO: 4, 22, 40, 62, 66, 69, 72 and 79.

[0125] For example, the anti-OX40 antibody or its antigen-binding portion described in this application includes a light chain variable region selected from the group shown in SEQ ID NO: 13, 31 and 49, or its light chain variable region CDR region satisfies at least one of the following sequences compared with the aforementioned sequences: a) binding to the same antigenic epitope, b) identity greater than 70%, 80%, 85%, 90% or 97%.

[0126] Furthermore, it includes a light chain variable region selected from the group consisting of sequences shown in SEQ ID NO: 13, 31 and 49, or a light chain variable region that, compared to the aforementioned sequences, satisfies at least one of the following: a) binds to the same antigenic epitope; b) has no more than 3 mutated amino acids.

[0127] Furthermore, it includes light chain variable regions selected from the group shown in SEQ ID NO: 13, 31, 49, 74 and 81.

[0128] The anti-OX40 antibody or its antigen-binding portion described in this application may be a whole antibody, a bispecific antibody, scFv, Fab, Fab', F(ab')2, or Fv. For example, when it is scFv, it may contain a linker peptide between the variable regions of its heavy and light chains, the sequence of which is shown in SEQ ID NO 1.

[0129] The anti-OX40 antibody or its antigen-binding portion described in this application may have a heavy chain constant region selected from IgG, IgM, IgE, IgD, and IgA. For example, its heavy chain constant region may be selected from IgG1, IgG2, IgG3, and IgG4. For example, its heavy chain constant region may be IgG1 or IgG4. For example, its heavy chain constant region may be selected from IgG1, wherein the heavy chain constant region of IgG1 contains mutations of S239D, I332E, and / or A330L. For example, the heavy chain constant region of the antibody may be selected from IgG1, wherein the heavy chain constant region of IgG1 contains mutations of S239D, I332E, and A330L. For example, the heavy chain constant region of the antibody may be selected from the amino acid sequences shown in any one of SEQ ID NO: 82-84.

[0130] The anti-OX40 antibody or its antigen-binding portion described in this application may have a light chain constant region that can be κ or λ. For example, its light chain constant region may contain an amino acid sequence as shown in SEQ ID NO: 85.

[0131] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants, including CDRs whose amino acid sequences have been substituted, deleted, and / or added one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; also covering its homologs, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR.

[0132] Furthermore, it should be noted that the antibody or antigen-binding fragment described in this application may contain heavy chain and / or light chain sequences with one or more conserved sequence modifications. "Conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the antibody-binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antigen-binding fragment described in this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the antibody or antigen-binding fragment described in this application may be replaced with other amino acid residues from the same side chain group. Those skilled in the art will know that some conserved sequence modifications do not cause the antigen to lose its binding ability. For example, see Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0133] The anti-OX40 antibody or its antigen-binding protein described in this application can be identified, screened, or characterized by various assays known in the art. For example, the antigen-binding activity of the antigen-binding protein or fusion protein of this application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0134] In this application, the antibody or antigen-binding fragment is specifically capable of binding to OX40. In some embodiments, the binding of the antibody or antigen-binding fragment to OX40 can be detected by an ELISA method. For example, the antigen-binding protein of this application can be expressed at EC50 concentrations less than or equal to about 0.010 μg / mL, less than or equal to about 0.009 μg / mL, less than or equal to about 0.008 μg / mL, less than or equal to about 0.007 μg / mL, less than or equal to about 0.006 μg / mL, less than or equal to about 0.005 μg / mL, less than or equal to about 0.004 μg / mL, less than or equal to about 0.003 μg / mL, less than or equal to about 0.002 μg / mL, and less than or equal to about 0.001 μg / mL. 50 The value is associated with OX40.

[0135] In this application, the antibody or antigen has enhanced ADCC activity. For example, the enhanced ADCC activity may involve mutation of the Fc region of the antibody. For example, the enhanced ADCC activity may involve mutation of the heavy chain constant region of the antibody. For example, mutations in the heavy chain constant region of the antibody, such as S239D, I332E, and / or A330L, have enhanced ADCC activity.

[0136] In some embodiments, the anti-OX40 antibody or its antigen-binding fragment is deglycosylated.

[0137] In some embodiments, the type of glycosylation modification includes N-position glycosylation or O-position glycosylation.

[0138] In some embodiments, the deglycosylation modification includes defucosylation modification.

[0139] In some embodiments, the deglycosylation modification site is located at the Fc terminus of the antibody.

[0140] In some embodiments, the defucosylation modification site includes Asn297.

[0141] The anti-OX40 antibody or its antigen-binding portion described in this application can be modified by defucosylation. The defucosylation modification may include removing or reducing the N-fucose at the asparagine (Asn297) position of the heavy chain of the antibody or its antigen-binding portion. The defucosylation modification of the antibody or its antigen-binding portion may include cell engineering methods, i.e., knocking out key genes (such as the FUT8 gene) responsible for fucose synthesis or transfer in host cells (e.g., CHO cells), causing the cells to naturally lack fucose in their secreted antibodies. The defucosylation modification of the antibody or its antigen-binding portion may also include in vitro enzymatic digestion, i.e., for purified antibodies (containing fucose), defucosylation is achieved by adding fucosidase in vitro to cleave the fucose in the Fc region. The defucosylation efficiency can be detected by UPLC or HPLC.

[0142] The defucosylated anti-OX40 antibody or its antigen-binding moiety described in this application can exhibit enhanced ADCC activity. The ADCC effect of the anti-OX40 antibody or its antigen-binding moiety described in this application can be detected using methods commonly used in the art. For example, the antibody to be tested can be co-incubated with target cells expressing the target antigen, and the degree of target cell lysis can be measured in the presence of effector cells (such as natural killer (NK) cells derived from human peripheral blood or FcγRⅢa positive cells). Target cell lysis can be quantified using lactate dehydrogenase (LDH) release assay, chromium-51 release assay, flow cytometry, or a fluorescence / luminescent substrate-based detection method. This allows the determination of the ADCC effect intensity of the antibody relative to the control antibody.

[0143] In some embodiments, the defucosylated anti-OX40 antibody or its antigen-binding portion may have enhanced therapeutic effects. In some embodiments, compared with unfucosylated anti-OX40 antibodies (e.g., DF004), defucosylated anti-OX40 antibodies (e.g., DF004) may further improve skin treatment efficacy, for example, by reducing skin scores by about 10%, 20%, 30%, 50%, or more; reducing dermatopathology scores by about 10%, 20%, 30%, 50%, or more; and reducing inflammatory cytokine (e.g., IL-6, IL-5, IL-4, IL-13) levels by about 10%, 20%, 30%, 50%, or more.

[0144] Nucleic acid molecules, vectors, cells, conjugates, and drug compositions

[0145] On the other hand, this application provides the use of isolated nucleic acid molecules in the preparation of pharmaceuticals for treating OX40-mediated diseases. For example, these diseases include inflammatory skin diseases.

[0146] The nucleic acid molecule described in this application may encode the anti-OX40 antibody or its antigen-binding fragment described in this application.

[0147] The nucleic acid molecule described in this application may contain a nucleic acid sequence capable of encoding the heavy chain variable region of the antibody described in this application, wherein the heavy chain variable region contains an amino acid sequence selected from the following group:

[0148] (1)SEQ ID NO: 5-7;

[0149] (2) SEQ ID NO: 23-25;

[0150] (3) SEQ ID NO: 41-43;

[0151] (4) A sequence that satisfies at least one of the following two conditions compared to the sequences (1)-(3) above: a) binds to the same antigenic epitope; b) has an identity greater than 70%, 80%, 85%, 90% or 97%.

[0152] Furthermore, the heavy chain variable region comprises an amino acid sequence selected from the following group:

[0153] SEQ ID NO: 4, 22 and 40, or a sequence that satisfies at least one of the following two conditions compared to the aforementioned sequences: a) binds to the same antigenic epitope, b) has an identity greater than 70%, 80%, 85%, 90% or 97%.

[0154] Furthermore, it includes a heavy chain variable region selected from the group consisting of sequences shown in SEQ ID NO: 4, 22 and 40, or a heavy chain variable region that, compared to the aforementioned sequences, satisfies at least one of the following: a) binds the same antigenic epitope; b) has no more than 3 mutated amino acids.

[0155] Furthermore, it includes heavy chain variable regions selected from the group shown in SEQ ID NO: 4, 22, 40, 62, 66, 69, 72 and 79.

[0156] The nucleic acid molecule described in this application further comprises a nucleic acid sequence capable of encoding an antibody heavy chain constant region; said heavy chain constant region is selected from IgG, IgM, IgE, IgD, and IgA. For example, its heavy chain constant region is selected from IgG1, IgG2, IgG3, and IgG4. For example, its heavy chain constant region is IgG1 or IgG4. For example, its heavy chain constant region is selected from IgG1, and said IgG1 heavy chain constant region contains mutations of S239D, I332E, and / or A330L.

[0157] The nucleic acid molecule described in this application may contain a nucleic acid sequence capable of encoding an antibody light chain variable region, wherein the light chain variable region comprises an amino acid sequence selected from the following group:

[0158] (1) SEQ ID NO: 14-16;

[0159] (2)SEQ ID NO: 32-34;

[0160] (3) SEQ ID NO: 50-52;

[0161] (4) A sequence that satisfies at least one of the following two conditions compared to the sequences (1)-(3) above: a) binds to the same antigenic epitope; b) has an identity greater than 70%, 80%, 85%, 90% or 97%.

[0162] Furthermore, the heavy chain variable region comprises an amino acid sequence selected from the following group:

[0163] SEQ ID NO: 13, 31 and 49, or sequences that satisfy at least one of the following two conditions compared to the aforementioned sequences: a) bind to the same antigenic epitope; b) have an identity greater than 70%, 80%, 85%, 90% or 97%.

[0164] Furthermore, it includes a light chain variable region selected from the group consisting of sequences shown in SEQ ID NO: 13, 31 and 49, or a light chain variable region that, compared to the aforementioned sequences, satisfies at least one of the following: a) binds to the same antigenic epitope; b) has no more than 3 mutated amino acids.

[0165] Furthermore, it includes light chain variable regions selected from the group shown in SEQ ID NO: 13, 31, 49, 74 and 81.

[0166] The nucleic acid molecule described in this application also includes a nucleic acid sequence capable of encoding the constant region of the antibody light chain. For example, the constant region of the light chain is κ-type or λ-type.

[0167] For example, the nucleic acid molecules described in this application may be generated or synthesized by: (i) in vitro amplification, for example by polymerase chain reaction (PCR); (ii) by clonal recombination; (iii) purification, for example by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, for example by chemical synthesis.

[0168] On the other hand, this application provides a carrier that can contain the nucleic acid molecule described in this application for use in the preparation of a medicament for treating OX40-mediated diseases. For example, the diseases include inflammatory skin diseases.

[0169] Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells to express the genetic material it carries within the host cells. The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. Furthermore, the vector may include components that facilitate its entry into the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0170] On the other hand, this application provides a cell that may contain the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain one or more of the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more types (e.g., two or more) of the nucleic acid molecules or vectors described in this application. For example, the vectors described in this application may be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi, or yeast cells. In some embodiments, the cell may be a bacterial cell (e.g., *Escherichia coli*), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NSO cells, or myeloma cells. The vectors described in this application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0171] On the other hand, this application also provides conjugates that may contain the antibody or antigen-binding fragments described in this application. For example, the conjugate may contain the anti-OX40 antibody or its antigen-binding portion described in this invention, as well as other bioactive substances, wherein the anti-OX40 antibody or its antigen-binding portion is conjugated directly or through a linker fragment to the other bioactive substances.

[0172] On the other hand, this application also provides pharmaceutical compositions that may comprise the antibody or antigen-binding fragment described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the conjugate described in this application, and optionally a pharmaceutically acceptable carrier.

[0173] In some embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0174] Methods and uses

[0175] On the other hand, this application provides the use of the antibody or antigen-binding fragment, the nucleic acid molecule, the carrier, the cell, the conjugate and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0176] On the other hand, this application also provides methods for preventing and / or treating diseases and / or conditions, which may include administering to a subject in need the antibody or antigen-binding fragment, the nucleic acid molecule, the carrier, the cell, the conjugate, and / or the pharmaceutical composition described in this application.

[0177] In this application, the use can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal application.

[0178] On the other hand, this application provides the use of the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate, and / or the pharmaceutical composition in the prevention and / or treatment of diseases and / or conditions.

[0179] On the other hand, this application provides the use of the antibody or antigen-binding fragment, the nucleic acid molecule, the carrier, the cell, the conjugate and / or the pharmaceutical composition in the preparation of a medicament for modulating an immune response.

[0180] On the other hand, this application provides the use of the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate, and / or the pharmaceutical composition in modulating an immune response.

[0181] On the other hand, this application also provides a method for modulating an immune response, the method of which may include administering to a subject in need the antibody or antigen-binding fragment described in this application, the nucleic acid molecule, the carrier, the cell, the conjugate, and / or the pharmaceutical composition described in this application.

[0182] The anti-OX40 antibody or antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the conjugate, and / or the pharmaceutical composition described in this application can modulate an immune response. For example, modulating the immune response may include upregulating the level of the immune response. For example, modulating the immune response may include downregulating the level of the immune response. For example, the immune response includes an inflammatory response. For example, the immune response includes increased MHC antigen expression. For example, the immune response includes the production and / or secretion of cytokines. For example, the immune response includes the activation of immune cells. For example, the immune response includes the proliferation and / or differentiation of immune cells. For example, the immune response includes the migration of immune cells. For example, the immune response includes the production and / or secretion of chemokines. For example, the immune response includes the production and / or secretion of antibodies.

[0183] Subjects and indications

[0184] On the other hand, the antibodies or antigen-binding fragments, nucleic acid molecules, vectors, cells, conjugates and / or pharmaceutical compositions described in this application can be used to prevent and / or treat diseases and / or conditions.

[0185] In this application, the diseases and / or conditions may include OX40-mediated diseases and / or conditions. In this application, the diseases and / or conditions may include diseases and / or conditions associated with OX40 expression. In this application, the diseases and / or conditions may include diseases and / or conditions associated with OX40 expression on T cells.

[0186] In this application, the OX40-mediated dermatitis may include allergy-related dermatitis.

[0187] In this application, the allergy-related dermatitis may include, but is not limited to, one or more of the following groups: urticaria, atopic dermatitis, atopic eczema, contact dermatitis, photodermatitis, white atrophy, allergic purpura, and allergic vasculitis.

[0188] In this application, the dermatitis may be atopic dermatitis.

[0189] In this application, the symptoms of atopic dermatitis include itching, erythema, edema, dryness, erosion / epidermal shedding, purulent exudation and crusting, lichenification, impaired skin barrier, and redness.

[0190] In this application, the characteristics of atopic dermatitis include: elevated levels of inflammatory cytokines, elevated levels of IgE antibodies, and / or elevated levels of vasoactive amines (such as histamine), enzymes, and lipid molecules (such as PGD2 and LTs).

[0191] The antibodies or antigen-binding fragments, nucleic acid molecules, vectors, cells, conjugates, and / or pharmaceutical compositions described in this application can be used to suppress at least one symptom of atopic dermatitis, including itching, erythema, edema, dryness, erosion / epidermal shedding, purulent exudation and crusting, lichenification, impaired skin barrier, and redness.

[0192] In some embodiments, the antibody or antigen-binding fragment, the nucleic acid molecule, the carrier, the cell, the conjugate, and / or the pharmaceutical composition described herein may be administered to a subject currently experiencing atopic dermatitis or symptoms of atopic dermatitis, and the administration of the antibody or antigen-binding fragment, the nucleic acid molecule, the carrier, the cell, the conjugate, and / or the pharmaceutical composition may inhibit one or more of these symptoms of atopic dermatitis.

[0193] In some embodiments, the antibody or antigen-binding fragment, the nucleic acid molecule, the carrier, the cell, the conjugate, and / or the pharmaceutical composition described herein may be administered to a subject who has previously developed atopic dermatitis or has previously experienced atopic dermatitis symptoms. The administration of the antibody or antigen-binding fragment, the nucleic acid molecule, the carrier, the cell, the conjugate, and / or the pharmaceutical composition may eliminate the development of atopic dermatitis in the patient or reduce the incidence of one or more atopic dermatitis symptoms.

[0194] In some embodiments, the diagnosis of atopic dermatitis described in this application may be performed in accordance with the Chinese Guidelines for the Diagnosis and Treatment of Atopic Dermatitis (Immunology Group of the Dermatology and Venereology Branch of the Chinese Medical Association, Collaborative Research Center for Atopic Dermatitis. Chinese Guidelines for the Diagnosis and Treatment of Atopic Dermatitis (2020 Edition) [J]. Chinese Journal of Dermatology, 2020, 53(02):81-88). In some embodiments, the diagnosis of atopic dermatitis described in this application may be performed in accordance with the "1+X" standard, i.e., the Chinese standard for atopic dermatitis.

[0195] In some embodiments, the atopic dermatitis described in this application can be diagnosed using multiple clinical methods, and the severity of atopic dermatitis can be quantified in practice and clinical trials using multi-item scoring tools. In some embodiments, the atopic dermatitis described in this application can be quantified using one or more of the following scoring tools: Scoring Alzheimer's Disease (SCORAD) score, Eczema Area and Severity Index (EASI) score, Investigator Global Assessment (IGA) score, Visual Analogue Scale (VAS) score for pruritus severity, Numerical Rating Scale for Pruritus (NRS), Patient Self-Examination Eczema Scale (POEM), Hospital Anxiety and Depression Scale (HADS), and Dermatology Quality of Life Scale (DLQI), etc.

[0196] The EASI assesses the severity of skin erythema, edema / infiltration / papules, scaling, lichenification, and pruritus in four body sites (head, trunk, arms, and legs), as well as the percentage of affected skin area. The percentage of affected area in each site is scored from 0 to 6. The total score ranges from 0 to 72. An EASI score of 7 or lower is considered mild, 8-21 moderate, 22-50 severe, and 51-72 very severe.

[0197] SCORAD uses different standards for individuals aged 2 years and older and those under 2 years old. SCORAD is calculated based on the area of ​​skin involved and the severity of redness, exudation, crusting, lichenification, and dryness, assessing the head, neck, arms, legs, forequarters, back, and genitals, with three scoring items: A, B, and C. The extent of skin lesions (A) divides the epidermis into multiple areas and assigns a corresponding percentage of body surface area to each area (9% for the head and neck, 9% for each upper limb, 18% for each lower limb, 18% for each front and back of the trunk, and 1% for the genital area), with 1 point awarded for every 1% of the lesion area. Clinical features (B) include erythema, edema / papules, exudation / crusting, epidermal peeling, lichenification, and dryness, each scored according to severity from 0 to 3 points. Patient self-report of pruritus and sleep quality (C) assesses pruritus and sleep loss over the previous 3 days. The SCORAD score is calculated as A / 5 + 7 × B / 2 + C, ranging from 0 to 103 points. Based on the score, the condition is classified as mild (0-24 points), moderate (25-50 points), or severe (>50 points).

[0198] Investigator Global Assessment (IGA): A rough assessment based on physicians' subjective judgment, allowing researchers to quickly evaluate disease severity at a given time point. This assessment categorizes severity into six levels (0-5).

[0199] Patient Self-Examination Rating Scale for Eczema (POEM): Patients assess the frequency and severity of seven symptoms (itching, sleep disturbance, bleeding, oozing, cracking, scaling, and dryness / roughness) over the past week. Each item is rated on a 5-point scale, corresponding to 0, 1, 2, 3, and 4 points. The score range is 0-28 points, with higher scores indicating more severe conditions.

[0200] The ADCT score (ADCT) includes six questions related to AD control, covering multiple key areas. It comprehensively assesses an AD patient's clinical signs, itching, sleep, anxiety / depression, and quality of life over the past week. A higher ADCT score indicates poorer AD control. It reflects both disease control at a specific point in time and changes in disease control over time, thus dynamically monitoring changes in the patient's condition. The score is patient-reported, assessing six AD symptoms over the past week, including overall symptom severity score, number of days with severe itching, irritability, sleep disturbances, and the degree of impact on daily life and mood. Each item is scored from 0 to 4 (most severe), with a total score of 0 to 24. A score <7 indicates disease control, and a score ≥7 indicates disease activity.

[0201] Peak pruritus NRS: This is a self-reported rating scale used by patients to assess pruritus over the past 24 hours. 0 points represent no pruritus, and 10 points represent the most severe pruritus they can imagine. Patients rate the most severe pruritus they have experienced in the past 24 hours.

[0202] Dermatology Quality of Life Index (DLQI): Patient-reported. The questionnaire contains 10 questions inquiring about the extent to which a skin condition has impacted a patient's daily life over the past week, including: the degree of itching, ulceration, pain, stinging, embarrassment, impact on shopping, housework, clothing choices, social and leisure activities, exercise, work or study, relationships with others, sex life, and the burden of seeking treatment. Each option is scored from 0 to 3, with irrelevant options scored as 0. The score ranges from 0 to 30, with higher scores indicating a greater impact on quality of life.

[0203] Childhood Dermatology Quality of Life Index (cDLQI): This index assesses the impact of skin diseases on a child's quality of life and includes 10 questions, such as: the degree of itching, pain, and burning; the degree of embarrassment or distress; the impact on social interactions, clothing, play, sports, schoolwork, and enjoyment of holidays; the degree of discrimination or bullying due to skin disease; the impact on sleep; and the degree of distress caused by skin disease treatment.

[0204] The Hospital Anxiety and Depression Scale (HADS) is a patient-reported scale used to assess a patient's anxiety and depression. It consists of two subscales: anxiety and depression. The mandatory questions on the anxiety scale include whether the patient feels tense, anxious, has experienced panic attacks, and is afraid of upcoming events. The mandatory questions on the depression scale include whether the patient is interested in previously enjoyed activities, can smile as usual, feels happy, and remains optimistic about the future. Each item is scored from 0 to 4. Higher scores indicate more severe anxiety or depression.

[0205] The European Five-Dimensional Health Inventory (EQ-5D) is used to assess patients' quality of life. It is a patient-reported assessment that includes five dimensions: mobility, self-care ability, daily activities, pain / discomfort, and anxiety / depression. Each dimension is rated on a three-point scale. In addition, patients must rate their health status using a visual analog scale (0-100 points).

[0206] Assessing the severity of Alzheimer's disease (AD) should not be limited to evaluating skin lesions alone; it should also consider the patient's self-reported symptom improvement. The IGA score is a holistic assessment by the physician, entirely based on subjective judgment, and accurate evaluation requires extensive experience in AD diagnosis and treatment. Unlike the EASI score, the SCORAD score further subdivides the scoring items. It includes not only assessments of clinical signs and manifestations but also patient self-reports of itching symptoms and sleep patterns.

[0207] While focusing on the disease of Alzheimer's disease (AD), their mental health should also be taken into account. The HADS scale is an optional scale for assessing anxiety / depression. It can also be discussed with a psychiatrist to select an appropriate and simple scale, enabling early identification of emotional abnormalities and providing intervention and counseling. This is also the future direction for comprehensive management of AD patients.

[0208] Objective assessment indicators, such as serum IgE testing and serological marker examinations, are somewhat correlated with the assessment of disease severity. Another commonly used indicator for assessing AD severity is thymic activation-regulated chemokine (TARC / CCL17), with serum TARC levels showing a correlation coefficient of 0.6-0.64 with disease severity, making it a specific laboratory indicator for assessing short-term AD severity. Other objective assessment indicators of AD severity include skin T-cell capture chemokines, serum E-selectin, macrophage-derived chemokines, lactate dehydrogenase, IL-4, and IL-13.

[0209] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0210] Example

[0211] The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of DF004 are shown in SEQ ID NO: 41, 42, and 70, respectively; the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 75-77, respectively; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 79; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 81; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 83; and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 85.

[0212] Due to significant differences in skin tissue structure among different species, this application uses the cynomolgus monkey, a primate, for experiments to better evaluate the therapeutic effect of DF004 on dermatitis. Cynomolgus monkeys share similar metabolic, functional, and structural biological characteristics with humans, making them an ideal model animal for studying related human diseases. Using cynomolgus monkeys as experimental subjects allows for a more realistic and effective evaluation of the actual efficacy of DF004 and an assessment of its feasibility in treating real human diseases.

[0213] Example 1: The therapeutic effect of DF004 on atopic dermatitis (AD) in cynomolgus monkeys.

[0214] Nine cynomolgus macaques, each weighing approximately 4-5 kg, were housed separately and randomly divided into three groups (model control group A, DF004 treatment group B, and no treatment group), with three macaques in each group. The model control groups corresponded to A1, A2, and A3, and the DF004 treatment groups corresponded to B1, B2, and B3, respectively. After 8 weeks of acclimatization and adaptation in the laboratory, atopic dermatitis was induced in the cynomolgus macaques. One day before induction (Day 0), the skin on the back and ears of the macaques was shaved, with a shaved area of ​​4cm × 6cm. The dermatitis induction procedures for the model control group and the treatment group are as follows:

[0215] On days 1, 2, and 3, apply 1% DNCB (dinitrochlorobenzene) evenly to the skin behind the ears and on the back of the cynomolgus monkeys.

[0216] From day 5 to day 27, 0.5% DNCB was applied evenly to the skin of the ears and back of the cynomolgus monkeys every two days. The model induction ended on day 27.

[0217] The DF004 treatment group also used the above induction procedure. After applying DNCB on day 1, the DF004 treatment group received intravenous DF004 once a week at a dose of 30 mg / kg for a total of four times.

[0218] The severity of skin inflammation in cynomolgus monkeys was observed and photographed daily. Figures 1A-1D show the degree of skin inflammation on the ears and backs of the monkeys. Figures 1A and 1C represent the control group without DF004 treatment, while Figures 1B and 1D represent the DF004 treatment group. As can be seen from the figures, in the control group, the skin of the cynomolgus monkeys gradually developed erythema, edema, dryness, lichenification, erosion / epidermal shedding, purulent exudation and crusting, skin barrier damage, and redness with increasing induction time, and the symptoms gradually worsened with the induction period. In contrast, in the DF004 treatment group, the skin redness and swelling of the cynomolgus monkeys were reduced, crusting decreased, and the inflammation improved, without progressing to severe dermatitis symptoms such as erosion and epidermal shedding.

[0219] Simultaneously, skin scores were assessed for each group of cynomolgus monkeys according to the criteria shown in Figure 3. Each symptom was scored as follows: 0 (normal), 1 (mild), 2 (moderate), 3 (severe), and 4 (extremely severe). The final score was the sum of the four symptoms, with a skin tissue inflammation score ranging from 0 to 16. As shown in the curves of Figure 2, the total skin score of the DF004 treatment group was significantly lower than that of the control group, indicating that the cynomolgus monkeys injected with DF004 generally showed better performance in terms of skin erythema, crusting, thickening, and dryness compared to the control group. Therefore, DF004 demonstrated a good therapeutic effect on atopic dermatitis in cynomolgus monkeys, significantly reducing the symptoms of atopic dermatitis on their skin.

[0220] Example 2: Toxicity and Dermatopathological Scoring Study of DF004 in the Treatment of Atopic Dermatitis (AD) in Cynomolgus Monkeys

[0221] The weight changes of the cynomolgus monkeys in each group were monitored regularly, and ear thickness was measured and recorded using electronic calipers. As shown in Figure 5 (weight ratio change curve) and Figure 6 (ear thickness change curve), the weight of the cynomolgus monkeys in the control group decreased significantly compared to the DF004 treatment group. The DF004 treatment group experienced weight loss in the first 14 days, gradually recovering to its pre-induction weight, demonstrating that the test animals showed low toxicity to DF004. Furthermore, the ear thickness of the control group was significantly increased relative to the DF004 treatment group, indicating that DF004 significantly alleviated the skin thickening symptoms of atopic dermatitis.

[0222] Based on the indicators in Table 1, the skin pathology scores of each group of cynomolgus monkeys were assessed. Each symptom was scored as follows: 0 (normal), 1 (mild), 2 (moderate), 3 (severe), and 4 (extremely severe). The final score was the sum of all symptoms. The skin tissue inflammation score ranged from 0 to 28 points. The scoring criteria are as follows:

[0223] 0 points: No abnormality, indicating that no lesions were found in the examined tissue section for this indicator.

[0224] 0.5 points: Mild, refers to very minor lesions that may only be visible under a microscope and have minimal impact on tissue structure and function.

[0225] 1 point: Mild, the lesion is relatively mild, but more obvious than mild, with slight changes in tissue structure.

[0226] 2 points: Moderate, with obvious lesions and some impact on tissue structure.

[0227] 3 points: Severe, with serious lesions and significant impact on tissue structure.

[0228] 4 points: Extremely severe, the lesion is extremely serious, and the tissue structure is greatly affected or completely destroyed.

[0229] As can be seen from the data in Table 1 and Figure 4, the skin pathology score of the DF004 group was significantly better than that of the model group, with an average score of only 6 points, which was significantly lower than the 13 points of the control group.

[0230] In conclusion, DF004 demonstrates excellent pathological efficacy in the treatment of atopic dermatitis (AD) in cynomolgus monkeys, exhibiting not only low therapeutic toxicity but also good therapeutic results in pathological scoring.

[0231] Table 1. Dermatopathological scores of DF004 in the treatment of atopic dermatitis (AD) in cynomolgus monkeys.

[0232] Example 3: Inflammatory cytokine levels after treatment of cynomolgus monkey atopic dermatitis (AD) with DF004.

[0233] A key characteristic of AD is the upregulation of cytokine levels, especially interleukin levels, in both diseased and non-diseased skin, suggesting that interleukin may contribute to the pathogenesis of AD to some extent.

[0234] On day 28, the cytokine levels in the skin of each group of cynomolgus monkeys were measured, including IL-6, IL-5, IL-4, and IL-13.

[0235] The levels of cytokines, including IL-6, IL-5, IL-4, and IL-13, in the serum of each group of cynomolgus monkeys were assessed using the LEGENDplex (Biolegend) Cytokine Kit (catalog number 740392) following the instructions in the manufacturer's manual.

[0236] The results of cytokine level detection are shown in Figure 7 and Table 2. The levels of IL-6, IL-5, IL-4, and IL-13 in the DF004 treatment group were significantly lower than those in the control group, indicating that DF004 can inhibit the production of inflammatory cytokines in cells, thereby inhibiting the occurrence of inflammation and treating atopic dermatitis of cynomolgus monkey skin.

[0237] Table 2. Inflammatory cytokine levels in cells.

[0238] Example 4: Preparation method of DF004-FKO

[0239] The following are the specific steps for producing fucose-free antibodies by knocking out the FUT8 gene in CHO cells using CRISPR technology:

[0240] (1) sgRNA design

[0241] sgRNA sequences were designed targeting the first and seventh exon sequences of the Fut8 genome in CHO cells. The complete sgRNA sequences were chemically synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the specific sequences are shown in Table 3.

[0242] Table 3: sgRNA sequences designed targeting exons 1 and 7 of the Fut8 gene

[0243] (2) Electroporation, cell line screening and antibody production

[0244] Take 2 μL (200 pmol) of sgRNA and 31 μL (200 pmol) of recombinant Cas9 protein, mix well, and incubate at room temperature for 10 min to prepare the RNP complex. Take CHO cells in logarithmic growth phase, centrifuge at 500g for 3 min, resuspend in CD CHO medium to 2×107 cells / mL, take 35 μL of CHO cells and gently mix with 35 μL of the above RNP complex, and incubate at room temperature for 10 min. Transfer the above system to an electroporation cuvette and perform electroporation according to the set parameters (voltage 250V, time 5ms, click count 1). After electroporation, transfer the cells to a 6-well cell culture plate and add 2 mL of [unspecified substance]. TransMaxC1 medium was used for culture at 37°C and 5% CO2. After 48 hours, lentil lectin (LCA) was added to a final concentration of 100 μg / mL, and the cells were cultured under pressure for 7 days. Transfection efficiency was detected by flow cytometry. After two rounds of limiting dilution, CHO cell lines with Fut8 gene knockout and defucose modification were identified and screened. Antibodies were produced using the screened CHO cell lines, and the defucose modified antibody was named DF004-FKO.

[0245] Example 5: Therapeutic effect of DF004-FKO on atopic dermatitis (AD) in cynomolgus monkeys.

[0246] The induction procedure and skin scoring criteria are the same as in Example 1.

[0247] Both the DF004 treatment group and the DF004-FKO treatment group received intravenous administration of DF004 once a week after applying DNCB on day 1, with each dose being 30 mg / kg, for a total of four administrations.

[0248] As shown in Figure 8, both the DF004 treatment group and the DF004-FKO treatment group showed good therapeutic effects on atopic dermatitis in cynomolgus monkeys, significantly reducing the symptoms of atopic dermatitis on the skin of cynomolgus monkeys. Compared with the DF004 treatment group, the DF004-FKO treatment group had a lower score, indicating better long-term therapeutic effects.

Claims

1. Use of an anti-OX40 antibody or its antigen-binding fragment in the preparation of a medicament for treating dermatitis, wherein the anti-OX40 antibody or its antigen-binding fragment comprises a CDR region selected from any of the following groups: 1) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 5-7, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 14-16, respectively. 2) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 23-25, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 32-34, respectively. 3) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 41-43, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 50-52, respectively. 4) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 63, 42, and 43, respectively, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 50-52, respectively. 5) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 41, 67, and 43, respectively, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 50-52, respectively. 6) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 41, 42, and 70, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 50-52, respectively. 7) The sequences of its heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 63, 67, and 70, respectively, and the sequences of its light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 75-77, respectively. 8) The sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 41, 42, and 70, respectively, and the sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 75-77, respectively.

2. The use according to claim 1, wherein the anti-OX40 antibody or its antigen-binding fragment comprises a heavy chain variable region of the following group, the sequences of which are shown in SEQ ID NO: 4, 22, 40, 62, 66, 72 and 79.

3. The use according to any one of claims 1-2, wherein the anti-OX40 antibody or its antigen-binding fragment comprises a light chain variable region of the following group, the sequences of which are shown in SEQ ID NO: 13, 31, 49, 74 and 81.

4. The use according to any one of claims 1-3, wherein the anti-OX40 antibody or its antigen-binding fragment comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 79 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:

81.

5. The use according to any one of claims 1-4, wherein the anti-OX40 antibody or its antigen-binding fragment is a whole antibody, a bispecific antibody, scFv, Fab, Fab', F(ab')2 or Fv.

6. The use according to any one of claims 1-5, wherein when the anti-OX40 antibody or its antigen-binding fragment is scFv, a linker peptide may be present between the variable regions of its heavy chain and light chain.

7. The use according to any one of claims 1-6, wherein the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment is selected from IgG, IgM, IgE, IgD and IgA.

8. The use according to any one of claims 1-7, wherein the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment is selected from IgG1 or IgG4.

9. The use according to any one of claims 1-8, wherein the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment is selected from IgG1, and the heavy chain constant region of the IgG1 contains mutations of S239D, I332E and / or A330L.

10. The use according to any one of claims 1-9, wherein the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO: 82-84.

11. The use according to any one of claims 1-10, wherein the light chain constant region of the anti-OX40 antibody or its antigen-binding fragment is κ or λ.

12. The use according to any one of claims 1-11, wherein the light chain constant region of the anti-OX40 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:

85.

13. The use according to any one of claims 1-12, wherein the heavy chain constant region of the anti-OX40 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 83, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

83. The amino acid sequence shown in NO:

85.

14. The use according to any one of claims 1-13, wherein the anti-OX40 antibody or its antigen-binding fragment is deglycosylated.

15. The use according to any one of claims 1-14, wherein the type of glycosylation modification includes N-position glycosylation or O-position glycosylation.

16. The use according to any one of claims 1-15, wherein the deglycosylation modification comprises defucosylation modification.

17. The use according to any one of claims 1-16, wherein the deglycosylation modification site is located at the Fc terminus of the antibody.

18. The use according to claim 17, wherein the defucosylation modification site comprises Asn297.

19. The use according to any one of claims 1-18, wherein the medicament comprises a nucleic acid molecule encoding an anti-OX40 antibody or an antigen-binding fragment thereof as described in any one of the preceding claims.

20. The use according to claim 19, wherein the drug comprises a carrier containing the nucleic acid molecule.

21. The use according to any one of claims 19-20, wherein the drug comprises a cell containing the nucleic acid molecule or the carrier.

22. The use according to any one of claims 1-21, wherein the medicament comprises a conjugate containing the anti-OX40 antibody or an antigen-binding fragment thereof.

23. The use according to any one of claims 1-22, wherein the medicament comprises a pharmaceutical composition comprising an anti-OX40 antibody or an antigen-binding fragment thereof as described in any one of the preceding claims, a nucleic acid molecule as described in the preceding claims, a carrier as described in the preceding claims, a cell as described in the preceding claims, and / or a conjugate as described in the preceding claims, and a pharmaceutically acceptable carrier.

24. The use according to any one of claims 1-23, wherein the dermatitis includes OX40-mediated dermatitis.

25. The use according to claim 24, wherein the OX40-mediated dermatitis includes allergy-related dermatitis.

26. The use according to claim 25, wherein the allergy-related dermatitis includes, but is not limited to, one or more of the following: urticaria, atopic dermatitis, atopic eczema, contact dermatitis, photodermatitis, white atrophy, allergic purpura, and allergic vasculitis.

27. The use according to any one of claims 24-26, wherein the dermatitis is atopic dermatitis (AD).

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