Antibodies or antigen-binding fragments that bind OX40

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that bind to OX40, and uses of the antibodies and antigen-binding fragments in treating diseases such as cancer. The antibodies or antigen-binding fragments include a heavy chain variable region, the heavy chain variable region including one or more CDRs having an amino acid sequence selected from SEQ ID NO: 1-12.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to an antibody or antigen-binding fragment binding to OX40, a nanobody binding to OX40, and uses thereof. Background Art

[0002] Immunotherapy is currently considered one of the main alternative approaches for cancer treatment [1–3] In particular, immunotherapy works by inhibiting or activating immune checkpoint molecules, such as the PD-1 / PD-L1 pathway (programmed cell death protein 1 / programmed cell death 1 ligand 1). [4–6] , CTLA-4 (cytotoxic T lymphocyte antigen 4) [7,8] , LAG-3 (lymphocyte activation gene) [9] OX40

[10] 、TIM-3 (T cell immunoglobulin and mucin domain 3)

[11] 、KIR (killer cell immunoglobulin-like receptor)

[12] and TIGIT (T cell immunoglobulin and ITIM domain)

[13] To reverse tumor immune escape.

[0003] OX40, also known as CD134, TNFRSF4 or ACT35, is a membrane protein expressed in CD4+ and CD8+ T cells, as well as neutrophils and NK cells, and is a member of the tumor necrosis factor receptor / tumor necrosis factor superfamily. OX40 is a type I transmembrane glycoprotein with a molecular weight of approximately 50kD. Its extracellular region has 191 amino acid residues, including three complete and one slightly shorter cysteine-rich domain (CRDs). OX40 is a positive co-stimulatory molecule on the surface of T cells. It is not expressed on the surface of resting T cells, but is highly expressed 24-72 hours after T cell activation. It binds to its ligand OX40L (also known as CD252 or TNFSF4) to transmit co-stimulatory signals. OX40 / OX40L signaling plays a very important role in the activation, proliferation and inhibition of apoptosis of T cells. Studies have shown that OX40 stimulates T cells to secrete a large number of cytokines and differentiate into memory and effector T cells through multiple signaling pathways (such as the NF-κB pathway). [14-16] In non-small cell lung cancer, ovarian cancer, advanced gastric cancer, advanced colorectal cancer, melanoma and glioblastoma, high expression of OX40 in tumor-infiltrating lymphocytes is associated with a good prognosis [17-23]Targeted agonist anti-OX40 monoclonal antibody combined with immunotherapy can improve the survival rate of mice with glioblastoma, and the anti-tumor immune effect of T helper cell type 1 (Th1) is significantly enhanced; combined anti-PD-1 / OX40 monoclonal antibody treatment can significantly inhibit the tumor growth of mouse ovarian cancer, causing CD8+T cells in the spleen to produce high levels of IFN-γ and show antigen-specific cytolytic activity after stimulation with tumor antigens. [24-26] Furthermore, OX40 co-stimulation abolished the suppressive function of Foxp3+ Treg cells constitutively expressing OX40 [10,27-28] . It can be seen that OX40 activates effector T cells and inhibits regulatory T cells, positively regulating and stimulating tumor immunity. Based on these characteristics, the OX40 receptor is considered to be one of the promising targets for new tumor immunotherapy.

[0004] In addition to its ligand OX40L, several agonist antibodies for mediating OX40 activation have been developed and are currently in early clinical stages. For example, pogalizumab, also known as MOXR0916 or RG7888, is a humanized effector agonist IgG1 monoclonal antibody targeting OX40. It is currently in clinical use in combination with atezolizumab for the treatment of advanced solid tumors. Initial results show that the combination shows good compliance.

[28] In addition, agonist antibodies such as Medi0562, IBI101 and BGB-A445 have also entered the clinic and are used in various types of anti-tumor treatments. Nevertheless, there is still a need to further design and develop differentiated OX40 agonist antibodies with better functions to reduce the potential risks of clinical trials. Summary of the invention

[0005] In a first aspect of the present disclosure, an antibody and an antigen-binding fragment thereof that binds to OX40 are provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH), and the VH comprises one or more CDRs having an amino acid sequence selected from SEQ ID NOs: 1-12.

[0006] GSIFSVYV (SEQ ID NO: 1).

[0007] ITPFDDNT (SEQ ID NO: 2).

[0008] AADWEWPEYNY (SEQ ID NO: 3).

[0009] GFIFSAYF (SEQ ID NO: 4).

[0010] INSNDDIT (SEQ ID NO: 5).

[0011] AAWLGAENYGY (SEQ ID NO: 6).

[0012] GSILDSNL (SEQ ID NO: 7).

[0013] INSYDDNT (SEQ ID NO: 8).

[0014] AAQVFVGWPYTDQMHDY (SEQ ID NO:9).

[0015] GSIYDFDV (SEQ ID NO: 10).

[0016] INSFGDIT (SEQ ID NO: 11).

[0017] AADWHVLIQQVLGY (SEQ ID NO: 12).

[0018] In some embodiments, the VH comprises CDR1, CDR2 and CDR3, wherein:

[0019] The CDR1 has an amino acid sequence shown in SEQ ID NO: 1, 4, 7 or 10;

[0020] The CDR2 has an amino acid sequence shown in SEQ ID NO: 2, 5, 8 or 11; and

[0021] The CDR3 has the amino acid sequence shown in SEQ ID NO: 3, 6, 9 or 12.

[0022] In some embodiments, the VH comprises:

[0023] a) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively;

[0024] b) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively;

[0025] c) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively; or

[0026] d) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively.

[0027] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) refers to an antibody that binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less affinity (K D ) binds to the antigen. D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0028] As used herein, the term "antibody" refers to an immunoglobulin molecule that contains at least one antigen recognition site and can specifically bind to an antigen. The term "antigen" refers to a substance that can induce an immune response in the body and specifically bind to an antibody, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination of the above substances. The binding of an antibody to an antigen is mediated by the interaction formed between the two, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The area on the surface of an antigen that binds to an antibody is an "antigenic determinant" or "epitope."

[0029] The term "antibody" as referred to in the present disclosure is understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies (e.g., bispecific antibodies) comprising at least two different antigen-binding domains. Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies from other sources. Antibodies may contain additional changes, such as unnatural amino acids, Fc effector function mutations, and glycosylation site mutations. Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of antibodies, and immunoglobulin molecules comprising any other modifications to antigen recognition sites, as long as these antibodies exhibit the desired biological activity. In other words, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing at least one antigen-binding domain.

[0030] As used herein, "variable region" (heavy chain variable region VH and light chain variable region VL) refers to the heavy chain and light chain parts that are directly involved in the binding of antibodies and antigens. Each VH and VL region is composed of three hypervariable regions or complementary determining regions (CDRs) and four framework regions (FRs) arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0031] As used herein, the term "CDR" refers to the complementary determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy chain and light chain, which are called CDR1, CDR2 and CDR3. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat et al. (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to antibody variable regions, but also provides residue boundaries that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Each complementary determining region can include amino acid residues of the "complementarity determining region" defined by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) found that Kabat Certain subsections within the CDRs adopt nearly identical peptide backbone conformations, despite having diversity at the amino acid sequence level. These subsections are referred to as L1, L2, and L3 or H1, H2, and H3, respectively, where "L" and "H" represent the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. There are other CDR boundary definitions that may not strictly follow one of the above systems, but will still overlap with Kabat CDRs. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use CDRs defined by Kabat or Chothia.

[0032] In some embodiments, the antibody is a monovalent, bivalent, or multivalent antibody.

[0033] In some embodiments, the antibody is a monospecific, bispecific, or multispecific antibody.

[0034] In some embodiments, the antibody is a bispecific antibody comprising a first antigen binding arm that binds OX40 and a second antigen binding arm that binds another antigen or another epitope on the OX40 molecule, wherein the first antigen binding arm comprises a heavy chain variable region (VH) as defined above.

[0035] In some embodiments, the antibody can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0036] According to the amino acid sequence of the constant region of the heavy chain of the antibody, immunoglobulins can be divided into 5 classes (isotypes): IgA, IgD, IgE, IgG and IgM, which can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. According to the amino acid sequence of the light chain, the light chain can be classified as a λ chain or a κ chain. The antibodies disclosed herein can be any of the above classes or subclasses.

[0037] In some embodiments, the antibodies of the present disclosure are selected from IgG, IgA, IgM, IgE, and IgD isotypes. In some embodiments, the antibodies of the present disclosure are IgG, for example selected from IgG1, IgG2, IgG3, and IgG4 subtypes.

[0038] As used herein, the term "antigen-binding fragment" includes, but is not limited to: a Fab fragment, which has VL, CL, VH and CH1 domains; a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; a Fd fragments having VH and CH1 domains; Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; Fv fragments and scFv having the VL and VH domains of a single arm of an antibody; dAb fragments consisting of either the VH domain or the VL domain; isolated CDR regions; F(ab')2 fragments, which are bivalent fragments comprising two Fab' fragments linked by a disulfide bridge at the hinge region; single-chain antibody molecules (e.g., single-chain Fv; scFv); "diabodies" having two antigen-binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain; "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen-binding regions; and modified forms of any of the foregoing that retain antigen-binding activity.

[0039] In some embodiments of the antibodies or antigen-binding fragments thereof of the present disclosure, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fd' fragment, a Fv fragment, a scFv fragment, a ds-scFv fragment, a dAb fragment, a single-chain fragment, a bivalent antibody, and a linear antibody.

[0040] In a second aspect of the present disclosure, a nanobody (also known as a single domain antibody or VHH) that binds to OX40 is provided, wherein the nanobody comprises one or more CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12.

[0041] In some embodiments, the Nanobody comprises CDR1, CDR2 and CDR3, wherein:

[0042] The CDR1 has an amino acid sequence shown in SEQ ID NO: 1, 4, 7 or 10;

[0043] The CDR2 has an amino acid sequence shown in SEQ ID NO: 2, 5, 8 or 11; and

[0044] The CDR3 has the amino acid sequence shown in SEQ ID NO: 3, 6, 9 or 12.

[0045] In some embodiments of the Nanobodies of the present disclosure, the Nanobody comprises:

[0046] a) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively;

[0047] b) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively;

[0048] c) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively; or

[0049] d) CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively.

[0050] In some embodiments, the Nanobody comprises a heavy chain framework region, at least a portion of which is from at least one of a mouse antibody, a camel antibody, a human antibody, a primate antibody, or a mutant thereof.

[0051] In some embodiments, the Nanobody has an amino acid sequence as shown in any one of SEQ ID NOs: 13 to 16, or an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 13 to 16. For example, the Nanobody may have an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with any one of SEQ ID NOs: 13 to 16.

[0052] EVQLVESGGGLVQPGGSLRLSCAASGSIFSVYVMGWFRQAPGKGRELVAAITPFDDNTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAADWEWPEYNYWGQGTQVTVSS (SEQ ID NO: 13).

[0053] EVQLVESGGGLVQPGGSLRLSCAASGFIFSAYFMGWFRQAPGKGRELVAAINSND DITYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAWLGAENYGYWGQGTQVTVSS (SEQ ID NO: 14).

[0054] EVQLVESGGGLVQPGGSLRLSCAASGSILDSNLMGWFRQAPGKGRELVASINSYD DNTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAQVFVGWPYTDQM HDYWGQGTQVTVSS (SEQ ID NO: 15).

[0055] EVQLVESGGGLVQPGGSLRLSCAASGSIYDFDVMGWFRQAPGKGRELVAAINSF GDITYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAADWHVLIQQVLGYWGQGTQVTVSS (SEQ ID NO: 16).

[0056] Herein, the amino acid sequence shown in SEQ ID NO:13 corresponds to a Nanobody called OX40 BC3-1; the amino acid sequence shown in SEQ ID NO:14 corresponds to a Nanobody called OX40 BC3-4; the amino acid sequence shown in SEQ ID NO:15 corresponds to a Nanobody called OX40 BC3-6; and the amino acid sequence shown in SEQ ID NO:16 corresponds to a Nanobody called OX40BC3-7.

[0057] As will be appreciated by those skilled in the art, the relatedness between two amino acid sequences or between two nucleotide sequences can be described by the parameter "sequence identity". The percentage of sequence identity between two sequences can be determined, for example, using a mathematical algorithm. The percentage of sequence identity between two sequences can be determined, for example, using a mathematical algorithm. Non-limiting examples of such mathematical algorithms include the algorithm of Myers and Miller (1988) CABIOS 4:11-17, the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the method for searching homology of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and a modification of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, the algorithm described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. By using a program based on such a mathematical algorithm, a sequence comparison (i.e., alignment) for determining sequence identity can be implemented. The program can be suitably executed by a computer. Examples of such programs include, but are not limited to, CLUSTAL, ALIGN programs (Version 2.0) of the PC / Gene program and GAP, BESTFIT, BLAST, FASTA, and TFASTA of the Wisconsin genetics software package. The alignment using these programs can be implemented, for example, by using initial parameters.

[0058] For example, the Nanobody may have an amino acid sequence in which one or more amino acid modifications are made in the amino acid sequence shown in any one of SEQ ID NOs: 13 to 16. In some embodiments, the amino acid modification does not change the CDR sequence of the antibody, i.e., the amino acid modification is made in the framework region (FR) of the variable region.

[0059] In some embodiments, the one or several amino acid modifications refer to 1-10 amino acid modifications or 1-5 amino acid modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

[0060] In some embodiments, the amino acid modification is selected from the substitution, deletion, addition and / or insertion of an amino acid residue. In some embodiments, the amino acid modification is an amino acid substitution, such as a conservative substitution. Such conservative substitutions are preferably substitutions of an amino acid residue in the following groups (a) to (e) by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) negatively charged residues and their amides: Asp, Asn, Glu and Gln; (c) positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; (e) aromatic residues: Phe, Tyr and Trp.

[0061] In a third aspect of the present disclosure, an isolated nucleic acid molecule is provided, which encodes an antibody or antigen-binding fragment according to the first aspect of the present disclosure, or a nanobody according to the second aspect of the present disclosure. In some embodiments, the nucleic acid molecule is DNA. In other embodiments, the nucleic acid molecule is RNA.

[0062] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NOs: 17-20 or a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NOs: 17-20. For example, the nucleic acid molecule can have a nucleotide sequence having at least 85%, at least 90%, at least 95, at least 98% or at least 99% sequence identity with any one of SEQ ID NOs: 17-20.

[0063] GAGGTGCAGCTGGTGGAAAGCGGCGGAGGACTGGTGCAACCCGGCGGCTCT CTGAGACTGAGCTGTGCCGCCTCCGGCTCTATCTTTAGTGTTTATGTTATGGGCTGGTTCAGACAAGCCCCGGCAAGGGCAGAGAGCTGGTGGCTGCTATTACCCCGTTTG ATGATAATAACCTATTACCCCGACTCCGTGGAGGGAAGATTCACCATCTCTAGAGACAACGCCAAGAGGATGGTGTACCTCCAGATGAACTCTCTGAGAGCCGAGGACACAG CCGTGTATTACTGCGCCGCTGACTGGGAATGGCCGGAATATAATTATTGGGGACAAGGCACCCAAGTGACCGTGAGTCCC (SEQ ID NO: 17)

[0064] GAGGTGCAGCTGGTGGAAAGCGGCGGAGGACTGGTGCAACCCGGCGGCTCT CTGAGACTGAGCTGTGCCGCCTCCGGCTTTATCTTTAGTGCTTATTTTATGGGCTGGTTCAGACAAGCCCCCGGCAAGGGCAGAGAGCTGGTGGCTGCTATTAACTCGAATG ATGATATTACCTATTACCCCGACTCCGTGGAGGGAAGATTCACCATCTCTAGAGACAACGCCAAGAGGATGGTGTACCTCCAGATGAACTCTCTGAGAGCCGAGGACACAGC CGTGTATTACTGCGCCGCTTGGCTGGGTGCTGAAAACTATGGCTATTGGGGACAAGGCACCCAAGTGACCGTGAGCTCC(SEQ ID NO:18);

[0065] GAGGTGCAGCTGGTGGAAAGCGGCGGAGGACTGGTGCAACCCGGCGGCTCT CTGAGACTGAGCTGTGCCGCCTCCGGCAGTATCTTAGACTCTAATCTTATGGGCTGGTTCAGACAAGCCCCCGGCAAGGGCAGAGAGCTGGTGGCTAGTATTAACTCGTAT GATGATAATACCTATTACCCCGACTCCGTGGAGGGAAGATTCACCATCTCTAGAGACAACGCCAAGAGGATGGTGTACCTCCAGATGAACTCTCTGAGAGCCGAGGACACA GCCGTGTATTACTGCGCCGCTCAGGTTTTCGTTGGTTGGCCGTACACTGACCAGATGCATGACTATTGGGGACAAGGCACCCAAGTGACCGTGAGCTCC(SEQ ID NO:19)

[0066] GAGGTGCAGCTGGTGGAAAGCGGCGGAGGACTGGTGCAACCCGGCGGCTCT CTGAGACTGAGCTTGTGCCGCCTCCGGCAGTATCTATGACTTTGATGTTATGGGCTG GTTCAGACAAGCCCCCGGCAAGGGCAGAGAGCTGGTGGCTGCTATTAACTCGTTTGGCGATATTACCTATTACCCCGACTCCGTGGAGGGAAGATTCACCATCTCTAGAGACAACGCCAAGAGGATGGTGTACCTCCAGATGAACTCTCTGAGAGCCGAGGACACAGCCGTGTATTACTGCGCCGCTGACTGGCATGTTCTGATCCAGCAGGTTCTTGGTTAT TGGGGACAAGGCACCCAAGTGACCGTGAGCTCC(SEQ IDNO:20)

[0067] The nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 17 encodes a Nanobody having the amino acid sequence shown in SEQ ID NO: 13; the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 18 encodes a Nanobody having the amino acid sequence shown in SEQ ID NO: 14; the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 19 encodes a Nanobody having the amino acid sequence shown in SEQ ID NO: 15; the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 20 encodes a Nanobody having the amino acid sequence shown in SEQ ID NO: 16.

[0068] In a fourth aspect of the present disclosure, a nucleic acid construct or vector, such as an expression vector, is provided, which includes a nucleic acid molecule according to the third aspect of the present disclosure. In some embodiments, the vector is a eukaryotic expression vector. In other embodiments, the vector is a prokaryotic expression vector.

[0069] As used herein, "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, and then the genetic material elements it carries are expressed in the host cell. Vectors are recognized by those skilled in the art and include but are not limited to: (1) plasmids; (2) phagemids; (3) cosmids; (4) artificial chromosomes, such as yeast artificial chromosomes, bacterial artificial chromosomes or P1-derived artificial chromosomes; (5) bacteriophages such as λ phage or M13 phage and (6) animal viruses, such as retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, poxviruses, and baculoviruses. A vector may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes; in addition, the vector may also contain a replication initiation site.

[0070] In a fifth aspect of the present disclosure, a cell is provided, which comprises the nucleic acid molecule according to the third aspect of the present disclosure or the nucleic acid construct or vector, such as an expression vector, according to the fourth aspect of the present disclosure.

[0071] In a sixth aspect of the present disclosure, an antibody conjugate is provided, comprising an antibody or antigen-binding fragment or nanobody that binds to OX40 of the present disclosure, wherein the antibody or antigen-binding fragment or nanobody is conjugated to an optional therapeutic agent, diagnostic agent or imaging agent (e.g., a cytotoxic molecule, a radioisotope, a fluorescent marker, a luminescent substance, a chromogenic substance or an enzyme). In some embodiments, the therapeutic agent may be a cytotoxic molecule, such as a small molecule compound.

[0072] In a seventh aspect of the present disclosure, a pharmaceutical composition is provided, which comprises an antibody or antigen-binding fragment, nanobody or antibody conjugate binding to OX40 of the present disclosure, and a pharmaceutically acceptable carrier, excipient or diluent.

[0073] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. As used herein, the phrase "pharmaceutically acceptable carrier, excipient and / or diluent" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid or solvent encapsulating material, which is involved in maintaining the stability, solubility or activity of the antibodies or antigen-binding fragments thereof of the present disclosure.

[0074] The compositions of the present disclosure can be formulated for administration to a subject in solid, liquid or gel form. For example, the compositions of the present disclosure can be formulated for parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection, as, for example, a sterile solution or suspension or a sustained release formulation.

[0075] In some embodiments, the composition further comprises one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents for radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer.

[0076] As used herein, the term "chemotherapeutic agent" refers to any chemical agent that has therapeutic usefulness in treating a disease characterized by abnormal cell growth. Chemotherapeutic agents as used herein include chemical agents and biological agents. These agents function to inhibit cellular activities that cancer cells rely on for continued survival. Classes of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and various anti-neoplastic drugs.

[0077] In an eighth aspect of the present disclosure, a method of treating cancer in a subject is provided, the method comprising the step of administering to the subject an effective amount of an antibody or antigen-binding fragment, nanobody, antibody conjugate or pharmaceutical composition of the present disclosure that binds to OX40.

[0078] As used herein, the term "treatment" refers to therapeutic treatment, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a condition associated with a disease or condition. The term "treatment" includes reducing or alleviating at least one side effect or symptom of a disease or condition. If one or more symptoms or clinical markers are reduced, the treatment is usually "effective". Alternatively, if the progression of the disease is reduced or stopped, the treatment is "effective", that is, "treatment" includes not only the improvement of symptoms, but also the cessation of the progression or deterioration of symptoms expected in the absence of treatment, at least slowing down. Beneficial or desired clinical results include, but are not limited to, alleviating one or more symptoms, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease state, delaying or slowing the progression of the disease, improving or alleviating the disease state, and alleviating (whether partially or completely), whether detectable or undetectable.

[0079] As used herein, the terms "subject", "patient" and "individual" are used interchangeably herein and refer to animals, such as humans. The term subject also includes "non-human mammals", such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs and non-human primates. In preferred embodiments, the subject is a human subject.

[0080] Specific examples of cancer include, but are not limited to, basal cell carcinoma, bile duct cancer; bladder cancer; bone cancer; breast cancer; peritoneal cancer; cervical cancer; bile duct cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; glioblastoma; liver cancer; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; B cell lymphoma; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia, etc.

[0081] In a preferred embodiment, the cancer is selected from colorectal cancer and glioblastoma.

[0082] In some embodiments of the above methods, the method further comprises the step of administering one or more additional therapies. For example, in some embodiments, the therapy is selected from chemotherapy, radiotherapy, immunotherapy and surgical therapy.

[0083] In a ninth aspect of the present disclosure, an antibody or antigen-binding fragment, nanobody, antibody conjugate or pharmaceutical composition binding to OX40 of the present disclosure is provided for use in treating cancer in a subject. In a preferred embodiment, the cancer is selected from colorectal cancer and glioblastoma.

[0084] In a tenth aspect of the present disclosure, there is provided a use of an antibody or antigen-binding fragment, nanobody, antibody conjugate or pharmaceutical composition that binds to OX40 of the present disclosure in the preparation of a medicament for treating cancer in a subject. In a preferred embodiment, the cancer is selected from colorectal cancer and glioblastoma.

[0085] In an eleventh aspect of the present disclosure, a kit for detecting OX40 is provided, comprising the antibody or antigen-binding fragment, nanobody or antibody conjugate that binds to OX40 of the present disclosure.

[0086] In a twelfth aspect of the present disclosure, there is provided use of the antibody or antigen-binding fragment, nanobody or antibody conjugate binding to OX40 of the present disclosure in the preparation of a kit for detecting OX40.

[0087] Further aspects and advantages will be described below, at least part of which will be apparent from the following description of the accompanying drawings, and / or will be apparent to one of ordinary skill in the art from the embodiments described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 The results of FACS analysis of human OX40 protein expression in L929-hOX40 cell line;

[0089] Figure 2 The results of FACS analysis of human OX40 protein expression in Jurkat-hOX40 cell line;

[0090] Figure 3 The results of FACS detection of the binding reaction between anti-OX40 nanobody and L929-hOX40 cell line;

[0091] Figure 4 The results of FACS detection of the binding reaction between anti-OX40 nanoantibodies and Jurkat-hOX40 cell lines;

[0092] Figure 5 The results of using the luciferase method to detect the expression level of NFkB RE-Luciferase in Jurkat / NFkB-Luc-hOX40 stable cell lines after PMA / Ionomycin stimulation;

[0093] Figure 6 The results of FACS detection of the expression level of hOX40 in Jurkat / NFkB-Luc-hOX40 stably transfected cell line;

[0094] Figure 7 The results of FACS detection of hCD32b expression level in L929-hCD32b stable cell line;

[0095] Figure 8 The results of the NFkB reporter gene experiment were used to detect the activation of the NFkB signaling pathway by anti-OX40 nanoantibodies;

[0096] Fig. 9 To use the NFkB reporter gene method to detect the results of the competition between anti-OX40 nanoantibodies and hOX40L for binding to human OX40 protein in Jurkat / NFkB-Luc-hOX40 stably transfected cell lines;

[0097] Fig.10 The results of FACS detection of the competition between OX40 nanobody and hOX40L for binding to human OX40 protein in L929-OX40 cell line;

[0098] Fig.11The results are as follows: FACS was used to detect the expression level of OX40 on the surface of T cells after PHA activation;

[0099] Fig.12 Based on the PBMC activation experiment, FACS was used to detect the binding reaction between anti-OX40 nanoantibodies and hOX40 on the surface of T cells;

[0100] Fig.13 The results of FACS detection of the binding of anti-OX40 nanobodies to CHO-S cells transiently transfected with different epitopes of human OX40. DETAILED DESCRIPTION

[0101] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0102] Example 1 Preparation of OX40 VHH-Fc Nanobody

[0103] 1. Screening of phage antibody library

[0104] (1) OX40-mFc protein (30 μg / mL dissolved in PBS, 100 μL / well) was coated on a 96-well Nunc Maxisorp immunoplate at 4°C overnight;

[0105] (2) Block with 2% milk (dissolved in PBS) at room temperature for 2 h, and then wash 2-4 times with PBST;

[0106] (3) Take about 10 13 Phages (dissolved in 2% milk; PBST) were added to 4 coated wells and incubated at room temperature with shaking for 2 h.

[0107] (4) Wash the coated plate 10 times with PBST, then add 100 μL of 100 mM HCl to each well. After elution at room temperature for 5 min, aspirate the eluate into an EP tube and add 1 / 8 volume of 1.0 M Tris-HCl (pH 11) for neutralization;

[0108] (5) Infect 3 mL of competent E. coli SS320 in the logarithmic growth phase (OD600 not exceeding 1.0) with the eluted phage. Cultivate at 37°C, 220 rpm for 30 min, then take out 200 μL of the infection product for storage. Add M13KO7 helper phage to the remaining bacterial solution (final concentration to 10 10 / mL) and continue culturing for 1h.

[0109] (6) Transfer the above culture solution to 50 mL 2YT medium (Carb + , Kan + ) at 37°C, 220 rpm overnight.

[0110] (7) Repeat steps (1) to (6) to perform the second and third rounds of screening.

[0111] (8) After three rounds of screening, the second and third rounds of infection products were spread on LB / Carb + On plates, cultured overnight at 37°C;

[0112] (9) The next day, the bacteria were selected to a concentration of 10 10 2YT medium (Carb + ) and cultured overnight.

[0113] (10) The binding of phage to OX40-mFc in the culture supernatant of the monoclonal clone was identified by ELISA, and positive clones were selected for Sanger sequencing.

[0114] Through three batches of repeated screening (three rounds of screening for each batch), a total of four different positive clone sequences with high binding activity to OX40-mFc were obtained (see Table 1).

[0115] Table 1. Binding activity and sequences of antibody clones

[0116]

[0117]

[0118] 2. VHH-Fc antibody expression and purification

[0119] The nucleotide sequences of the four positive clones VHH in the phage supernatant were amplified by PCR and expressed in 5% paraformaldehyde with In- HD Cloning Kit (purchased from Takara) was fused to CMV-12GS-FC vector with coding linker and human IgG FC fragment (hFC). Subsequently, the constructed plasmid was used for transient protein secretion expression and expansion culture in ExpiCHO cells and matching transfection kit (purchased from Gibco). After 7 days, the cell culture fluid was collected and centrifuged at 4000rpm for 15 minutes to remove cell impurities and other components to obtain the culture supernatant containing the extracellular region of OX40 protein.

[0120] The harvested OX40 supernatant was loaded onto a Protein A affinity chromatography column (purchased from GE), and the change in ultraviolet absorption value (A280nm) was detected simultaneously, and eluted with 20mM sodium acetate (pH3.5) to obtain the above-mentioned 4 humanized nanobodies targeting OX40.

[0121] Example 2 Specific binding of OX40 VHH-Fc antibody to cell lines expressing OX40

[0122] 2.1 Construction of stable cell lines expressing OX40 protein

[0123] The nucleotide sequence encoding the amino acid sequence of the full-length human OX40 protein (the sequence is derived from the NCBI database, the protein sequence number is NP_003318.1) was cloned into the PCMV3-Hygromycin vector to prepare a plasmid carrying the human OX40 protein coding sequence (herein referred to as PCMV3-hOX40-Hygro). The above plasmids were used to transfect L929 and Jurkat cell lines, respectively. The transfected L929 and Jurkat cells were selectively cultured in DMEM+10% FBS and RPMI1640+10% FBS culture fluids containing 500 μg / mL hygromycin, and the surviving cells were picked after one week. Cloning was performed in a 96-well culture plate by limiting dilution method. After about 2 weeks, some monoclones were selected and expanded into 24-well plates, and then expanded into 6-well plates after about 3 to 4 days. The amplified monoclones were screened by flow cytometry using anti-human OX40 antibodies. The cell lines with good growth, high fluorescence intensity and monoclonal were selected to continue to expand the culture (the concentration of hygromycin was halved) and cryopreserved in liquid nitrogen. The L929 and Jurkat stable cell lines expressing human OX40 protein were obtained, which were called L929-hOX40 and Jurkat-hOX40 stable cell lines.

[0124] like Figure 1 and Figure 2 As shown in the results, the screened L929-hOX40 and Jurkat-hOX40 cell lines stably express human OX40.

[0125] 2.2 FACS detection of antibody binding to L929-hOX40 cells

[0126] The L929-hOX40 stable cell line constructed in Example 2.1 was expanded and cultured in a cell culture flask to 90% confluence. After washing with PBS, the cells were digested with 0.25% Trypsin (purchased from Thermofisher) to form a single cell suspension, neutralized with a culture medium containing 10% FBS, and counted. After centrifugation at 1000 rpm for 5 minutes, the cells were resuspended in FACS buffer (PBS + 1% FBS) to 2 × 10 6Cells / mL, 200 μL per well was added to a 96-well FACS culture plate, centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. A series of concentration gradients of anti-human OX40 test antibodies or isotype controls were added, 200 μL per well, and incubated at 4°C in the dark for 1 hour. After centrifugation at 1000 rpm for 5 minutes, 200 μL of PE fluorescently labeled anti-human FC secondary antibody (purchased from Invitrogen) was added to each well, and incubated at 4°C in the dark for 1 hour. Wash twice with FACS buffer, then add 200 μL of PBS buffer containing 1% paraformaldehyde per well to resuspend the cells, and detect and analyze with a FACS instrument (BD Accuri C6).

[0127] The results are as follows Figure 3 As shown in Table 2, anti-human OX40 antibodies BC3-1, BC3-4, BC3-6 and BC3-7 can specifically bind to hOX40 protein on the cell surface. The isotype control is human IgG, the positive control is MoxR-0916, and the MFI in the figure is the mean fluorescence intensity value of the measured cell population. The maximum mean fluorescence intensity value and EC50 value of the antibody binding to L929-hOX40 cells are shown in Table 2.

[0128] Table 2: Binding of antibodies to L929-hOX40 cell lines stably expressing OX40 protein

[0129]

[0130] 2.3 FACS detection of antibody binding to Jurkat-hOX40 cells

[0131] The Jurkat-hOX40 stable cell line constructed in Example 2.1 was expanded in a cell culture flask, and cells in the logarithmic growth phase were counted. The cells were centrifuged at 1000 rpm for 5 minutes and then resuspended in FACS buffer (PBS + 1% FBS) to 2×10 6 Cells / mL, 200 μL per well was added to a 96-well FACS culture plate. After centrifugation at 1000 rpm for 5 minutes, the supernatant was removed, and a series of concentration gradients of purified anti-human OX40 antibodies to be tested or isotype controls were added, 200 μL per well, incubated at 4°C in the dark for 1 hour, centrifuged at 1000 rpm for 5 minutes, 200 μL of PE fluorescently labeled anti-human FC secondary antibody (purchased from Invitrogen) was added per well, and incubated at 4°C in the dark for 1 hour. Wash twice with FACS buffer, add 200 μL of PBS buffer containing 1% paraformaldehyde per well to resuspend the cells, and detect and analyze with a FACS instrument (BD Accuri C6).

[0132] The results are as follows Figure 4As shown in the figure, the anti-human OX40 test antibodies BC3-1, BC3-4, BC3-6 and BC3-7 can specifically bind to the hOX40 protein on the cell surface. The isotype control is human IgG, the positive control is MoxR-0916, and the MFI in the figure is the mean fluorescence intensity value of the measured cell population. The maximum mean fluorescence intensity value and EC50 value of the antibody binding to the Jurkat-hOX40 cell line are shown in Table 3.

[0133] Table 3: Antibody binding to Jurkat-hOX40 cell lines stably expressing OX40 protein

[0134]

[0135]

[0136] Example 3OX40 VHH-Fc antibody activates NF-kB signaling pathway

[0137] 3.1 Construction of Jurkat / NFkB-Luc-hOX40 stable cell line

[0138] The pGL4.32[luc2P / NF-kB-RE / Hygro] plasmid (purchased from Promega) was transfected into Jurkat cells, cultured with RPMI1640 culture medium containing hygromycin and 10% FBS, and the Jurkat cell line Jurkat / NF-kB-Luc stably expressing NF-kB-RE-Luc was screened. The OX40 lentiviral plasmid was further constructed, and the lentivirus was packaged and infected into Jurkat-NF-kB-Luc cells. The infected cells were cultured with RPMI1640 culture medium containing hygromycin and 10% FBS, and the Jurkat cell line stably expressing NF-kB-RE-Luc and OX40 was screened, which was called Jurkat / NF-kB-Luc-hOX40.

[0139] Figure 5 The experimental results of NF-kB reporter gene are shown, indicating that the screened Jurkat / NF-kB-Luc-hOX40 cell line stably expresses NF-kB-RE luciferase. Figure 6 The FACS test results are shown, indicating that the screened Jurkat / NF-kB-Luc-hOX40 cell line stably expresses hOX40.

[0140] 3.2 Construction of L929-CD32b stable cell line

[0141] The L929 cell line was transfected with a plasmid carrying the hCD32b gene. The transfected cells were selectively cultured in DMEM + 10% FBS medium containing 600 μg / ml hygromycin, and the surviving cells were picked for cloning and subcloned in a 96-well culture plate using the limiting dilution method. After about 2 weeks, some monoclones were selected and expanded into 24-well plates, and then expanded into 6-well plates after about 3 to 4 days. The amplified clones were screened by flow cytometry using CD32b antibodies (purchased from Stemcell). Figure 7 shows the results of FACS detection, indicating that the screened L929-hCD32b cell line stably expresses hCD32b.

[0142] 3.3 NF-kB reporter gene experiment

[0143] The L929-hCD32b cells screened in Example 3.2 were expanded and cultured, and the cells were collected and resuspended in DMEM culture medium containing 10% FBS. 4 / well in a 96-well culture plate. After overnight, the supernatant was discarded and 5×10 4 The Jurkat / NF-kB-Luc-hOX40 cells screened in 3.1 were added to the cells at a cell density of 1 / well, and a series of concentration gradients of anti-human OX40 antibodies to be tested or isotype controls were added, and the cells were placed in a CO2 cell culture incubator and cultured for 5 hours. The luciferase content was determined using the ONE-Glo luciferase detection system (purchased from Promega).

[0144] The results are as follows Figure 8 As shown, the isotype control is human IgG, the positive control is MoxR-0916, and RLU represents the relative luminescent signal value. The results show that the anti-human OX40 antibodies BC3-1, BC3-4, BC3-6 and BC3-7 can significantly activate the NF-kB signaling pathway.

[0145] Table 4: Antibodies activate NF-kB signaling pathway

[0146] Antibody Maximum value (RLU) <![CDATA[EC 50 (pM)]]> Isotype Control 2650 / BC3-1 53365 209.2 BC3-4 55747 175.1 BC3-6 50273 162.4 BC3-7 48390 129.8 MoxR-0916 29821 216.9

[0147] Example 4 Inhibitory effect of anti-human OX40 antibodies on the binding of OX40 and OX40L proteins

[0148] 4.1 Detection of the inhibitory effect of anti-human OX40 antibody on the binding of OX40 and OX40L proteins by reporter gene assay

[0149] Jurkat / NF-kB-Luc-hOX40 cells constructed in Example 3.1 were used at 5×10 4The cells were added to a 96-well cell culture plate at a cell density of 100 μg / well. OX40L protein diluted to a concentration of 0.3 μg / ml and a series of gradient dilutions of anti-human OX40 antibody or isotype control were added to the cells at the same time. After incubation in a CO2 incubator for 5 hours, the luciferase content was determined. The results are shown in Figure 2. Fig. 9 As shown, the results showed that the anti-human OX40 antibodies BC3-1, BC3-4, BC3-6 and BC3-7 were able to inhibit the binding of OX40L to OX40, indicating that there was an epitope competition relationship between these antibodies and OX40L.

[0150] 4.2 FACS detection of the inhibitory effect of anti-human OX40 antibody on the binding of OX40 and OX40L proteins

[0151] The L929-hOX40 stable cell line constructed in Example 2.1 was expanded and cultured in a cell culture flask to 90% confluence. The cells were washed with PBS, digested with 0.25% Trypsin (purchased from Thermofisher) to form a single cell suspension, and counted after neutralization with a culture medium containing 10% FBS. The cells were centrifuged at 1000 rpm for 5 minutes and resuspended in FACS buffer (PBS + 1% FBS) to 2×10 6 Cells / mL. 200 μL was added to each well of a 96-well FACS culture plate, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. At the same time, 0.5 μg / ml anti-human OX40 antibody or isotype control and a series of gradient dilutions of OX40L protein were added, and incubated at 4°C in the dark for 1 hour. After centrifugation at 1000 rpm for 5 minutes, 200 μL of PE fluorescently labeled anti-human FC secondary antibody (purchased from Invitrogen) was added to each well, and incubated at 4°C in the dark for 1 hour. Wash twice with FACS buffer, add 200 μL of PBS buffer containing 1% paraformaldehyde to each well to resuspend the cells, and detect and analyze with a FACS instrument (BD Accuri C6).

[0152] The results are as follows Fig.10 The results showed that the anti-human OX40 antibodies BC3-1, BC3-4, BC3-6 and BC3-7 all competed with OX40L for binding to OX40, further indicating that there was an epitope competition relationship between these antibodies and OX40L.

[0153] Example 5 Anti-human OX40 antibody binds to OX40 on the surface of T cells

[0154] 5.1 Cryopreserved PBMC cells (purchased from AllCells) were cultured at 0.5×10 6The cells were revived at a seeding density of 10 cells / ml in RPMI 1640 culture medium containing 10% FBS, and 2μg / ml PHA and 2μg / ml IL-2 were added. The cells were incubated in an incubator for 48 hours and then collected. The cells were centrifuged at 1000 rpm for 5 minutes and resuspended in FACS buffer (PBS + 1% FBS) to 2×10 6 cells / mL, and 200 μL was added to each well of a 96-well FACS culture plate. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and 5 μL of APC fluorescently labeled anti-human OX40 antibody (purchased from Biolegend) was added to each well to detect the expression level of OX40 on the surface of T cells after T cells in PBMCs were activated by PHA.

[0155] The results are as follows Fig.11 As shown, resting T cells do not express OX40. After PHA activation, activated T cells express high levels of OX40.

[0156] 5.2 Cryopreserved PBMC cells (purchased from AllCells) were cultured at 0.5×10 6 The cells were revived at a seeding density of 10 cells / ml in RPMI 1640 culture medium containing 10% FBS, and 2μg / ml PHA and 2μg / ml IL-2 were added. The cells were placed in an incubator and incubated for 48 hours and then collected. The cells were centrifuged at 1000 rpm for 5 minutes and resuspended in FACS buffer (PBS + 1% FBS) to 3×10 6 cells / mL, and 200 μL was added to each well of a 96-well FACS culture plate. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and a series of gradient dilutions of anti-OX40 antibodies were added, and incubated at 4°C in the dark for 1 hour; after centrifugation at 1000 rpm for 5 minutes, 200 μL of PE fluorescently labeled anti-human FC secondary antibody (purchased from Invitrogen) was added to each well, and incubated at 4°C in the dark for 1 hour. Wash twice with FACS buffer, add 200 μL of PBS containing 1% paraformaldehyde to each well to resuspend the cells, and detect and analyze with a FACS instrument (BD Accuri C6).

[0157] The results are as follows Fig.12 As shown, the MFI in the figure is the mean fluorescence intensity value of the measured cell population. The results show that the anti-human OX40 antibodies BC3-1, BC3-4, BC3-6 and BC3-7 can specifically bind to the hOX40 protein on the surface of activated T cells in a concentration-dependent manner.

[0158] Example 6 Identification of Antigenic Epitopes of Anti-Human OX40 Antibodies

[0159] 6.1 Construction of plasmids expressing different domains of human OX40

[0160] Using the PCMV3 plasmid as the backbone, a truncated expression vector of OX40 (human) lacking the CRD1 domain was constructed, named PCMV3-SP-OX40(CRD2-3-4)(human); a truncated expression vector lacking CRD1 and CRD2 (human) was constructed, named PCMV3-SP-OX40(CRD3-4)(human); a chimeric expression vector lacking human CDR1 and CRD2, replacing human CRD3 with mouse CRD3, and containing the human CRD4 domain was constructed, named PCMV3-SP-OX40(CRD3mus-CRD4human); a mouse full-length OX40 expression vector was constructed, named PCMV3-OX40(Mus); and a human full-length OX40 expression vector was constructed, named PCMV3-OX40(human).

[0161] 6.2 Identification of Antigenic Epitopes of Anti-human OX40 Antibodies by FACS

[0162] Transient transfection of OX40 expression vector: After thawing CHO-S cells and passaged at least twice, cells were cultured at 1×10 6 The cells were seeded into 6-well plates at a density of 10 cells / well. The next day, the expression vectors expressing different domains of OX40 were transfected into CHO-S cells using the PEI transfection method. 24 hours after transfection, anti-human OX40 antibodies BC3-1, BC3-4, BC3-6, and BC3-7 were incubated with the transfected cells, and the binding of antibodies to cells was detected by FACS. Fig.13 As shown in the results, anti-human OX40 antibodies BC3-1, BC3-4, BC3-6, and BC3-7 bind to CHOS-OX40 (CRD2-3-4human) and CHOS-OX40 (human), but do not bind to CHOS, CHOS-OX40 (mus), CHOS-OX40 (CRD3-4human), and CHOS-OX40 (CRD3mus-CRD4human). The above results indicate that the antigenic epitopes of BC3-1, BC3-4, BC3-6, and BC3-7 with OX40 are located in CRD2.

[0163] The contents of all references cited throughout this application (including literature references, issued patents, published patent applications and concurrently pending patent applications) are hereby expressly incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly known to those of ordinary skill in the art.

[0164] According to the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the appended claims.

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Sequence Listing <110> BioDuro Technology (Beijing) Co., Ltd. <120> Antibodies or antigen-binding fragments that bind OX40 <130> C22P1764 <150> 202110480042X <151> 2021-04-30 <160> 20 <170> PatentIn Version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 <400> 1 Gly Ser Ile Phe Ser Val Tyr Val 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 <400> 2 Ile Thr Pro Phe Asp Asp Asn Thr 1 5 <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 <400> 3 Ala Ala Asp Trp Glu Trp Pro Glu Tyr Asn Tyr 1 5 10 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 <400> 4 Gly Phe Ile Phe Ser Ala Tyr Phe 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 <400> 5 Ile Asn Ser Asn Asp Asp Ile Thr 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 <400> 6 Ala Ala Trp Leu Gly Ala Glu Asn Tyr Gly Tyr 1 5 10 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 <400> 7 Gly Ser Ile Leu Asp Ser Asn Leu 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 <400> 8 Ile Asn Ser Tyr Asp Asp Asn Thr 1 5 <210> 9 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 <400> 9 Ala Ala Gln Val Phe Val Gly Trp Pro Tyr Thr Asp Gln Met His Asp 1 5 10 15 Tyr <210> 10 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH CDR1 <400> 10 Gly Ser Ile Tyr Asp Phe Asp Val 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH CDR2 <400> 11 Ile Asn Ser Phe Gly Asp Ile Thr 1 5 <210> 12 <211> 14 <212> PRT <213> Artificial sequence <220> <223> VH CDR3 <400> 12 Ala Ala Asp Trp His Val Leu Ile Gln Gln Val Leu Gly Tyr 1 5 10 <210> 13 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of nanobody <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Val Tyr 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45 Ala Ala Ile Thr Pro Phe Asp Asp Asn Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Trp Glu Trp Pro Glu Tyr Asn Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 14 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of nanobody <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ala Tyr 20 25 30 Phe Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asn Asp Asp Ile Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Trp Leu Gly Ala Glu Asn Tyr Gly Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 15 <211> 124 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of nanobody <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Asp Ser Asn 20 25 30 Leu Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45 Ala Ser Ile Asn Ser Tyr Asp Asp Asn Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gln Val Phe Val Gly Trp Pro Tyr Thr Asp Gln Met His Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 16 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of nanobody <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Tyr Asp Phe Asp 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Phe Gly Asp Ile Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Light Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Trp His Val Leu Ile Gln Gln Val Leu Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 17 <211> 354 <212> DNA <213> The snowstorm <220> <223> Connection license plate <400> 17 60. gaggtgcagc tggtggaag cggcggagga ctggtgcaac ccggcggctc tctgagactg agctgtgccg cctccggctc tatctttagt gtttatgtta tgggctggtt cagacaagcc cccggcaagg gcagagagct ggtggctgct attackcccgt ttgatgataa tacctattac cccgactccg tggagggag attcaccatc tctagagaca acgccaagag gatggtgtac 240 ctccagatga actctctgag agccgaggac acagccgtgt attactgcgc cgctgactgg gaatggccgg aatataatta ttggggacaa ggcacccaag tgaccgtgag ctcc 354 <210> 18 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence <400> 18 gaggtgcagc tggtggaaag cggcggagga ctggtgcaac ccggcggctc tctgagactg 60 agctgtgccg cctccggctt tatctttagt gcttatttta tgggctggtt cagacaagcc 120 cccggcaagg gcagagagct ggtggctgct attaactcga atgatgatat tacctattac 180 cccgactccg tggagggaag attcaccatc tctagagaca acgccaagag gatggtgtac 240 ctccagatga actctctgag agccgaggac acagccgtgt attactgcgc cgcttggctg 300 ggtgctgaaa actatggcta ttggggacaa ggcacccaag tgaccgtgag ctcc 354 <210> 19 <211> 372 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence <400> 19 gaggtgcagc tggtggaaag cggcggagga ctggtgcaac ccggcggctc tctgagactg 60 agctgtgccg cctccggcag tatcttagac tctaatctta tgggctggtt cagacaagcc 120 cccggcaagg gcagagagct ggtggctagt attaactcgt atgatgataa tacctattac 180 cccgactccg tggagggaag attcaccatc tctagagaca acgccaagag gatggtgtac 240 ctccagatga actctctgag agccgaggac acagccgtgt attactgcgc cgctcaggtt 300 ttcgttggtt ggccgtacac tgaccagatg catgactatt ggggacaagg cacccaagtg 360 accgtgagct cc 372 <210> 20 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide Sequence <400> 20 gaggtgcagc tggtggaaag cggcggagga ctggtgcaac ccggcggctc tctgagactg 60 agctgtgccg cctccggcag tatctatgac tttgatgtta tgggctggtt cagacaagcc 120 cccggcaagg gcagagagct ggtggctgct attaactcgt ttggcgatat tacctattac 180 cccgactccg tggagggaag attcaccatc tctagagaca acgccaagag gatggtgtac 240 ctccagatga actctctgag agccgaggac acagccgtgt attactgcgc cgctgactgg 300 catgttctga tccagcaggt tcttggttat tggggacaag gcacccaagt gaccgtgagc 360 tcc 363

Claims

1. A Nanobody that binds to OX40, wherein the Nanobody comprises: a) CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively; b) CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively; c) CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively; or d) CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NOs: 10, 11 and 12, respectively.

2. The Nanobody according to claim 1, wherein the Nanobody comprises a heavy chain framework region, at least a portion of which is derived from at least one of a mouse antibody, a camel antibody, and a primate antibody.

3. The Nanobody according to claim 2, wherein at least a portion of the heavy chain framework region is from a human antibody.

4. The Nanobody according to claim 2, wherein the amino acid sequence of the Nanobody is as shown in any one of SEQ ID NOs: 13-16.

5. An isolated nucleic acid molecule encoding the Nanobody according to any one of claims 1 to 4. The nucleic acid molecule according to claim 5 , wherein the nucleic acid molecule is DNA.

7. The nucleic acid molecule according to claim 5, wherein the nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NOs: 17-20.

8. An expression vector comprising the nucleic acid molecule according to any one of claims 5 to 7.

9. The expression vector according to claim 8, wherein the expression vector is a eukaryotic cell expression vector.

10. A cell comprising the isolated nucleic acid molecule of any one of claims 5-7 or the expression vector of any one of claims 8-9.

11. An antibody conjugate comprising the Nanobody according to any one of claims 1 to 4 conjugated to a therapeutic agent, a diagnostic agent or an imaging agent.

12. The antibody conjugate of claim 11, wherein the therapeutic agent is a cytotoxic molecule.

13. A pharmaceutical composition comprising the Nanobody according to any one of claims 1 to 4 or the antibody conjugate according to claim 11 or 12, and a pharmaceutically acceptable excipient.

14. Use of the Nanobody of any one of claims 1 to 4, the antibody conjugate of claim 11 or 12, or the pharmaceutical composition of claim 13 in the preparation of a medicament for treating cancer, wherein the cancer is selected from colorectal cancer and glioblastoma.

15. A kit for detecting OX40, comprising the Nanobody according to any one of claims 1 to 4 or the antibody conjugate according to claim 11 or 12.

16. Use of the Nanobody according to any one of claims 1 to 4 or the antibody conjugate according to claim 11 or 12 in the preparation of a kit for detecting OX40.

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