Anti-ox40 single-domain antibody and use thereof

Abstract

A specific single-domain antibody targeting human OX40, having three complementarity determining regions (CDRs) derived from VHH chain amino acid sequences as shown in SEQ ID NOs: 1-10. Provided are a coding sequence, an expression vector and a host cell for the single-domain antibody, a production method, an immunoconjugate, a use, and a pharmaceutical composition. The single-domain antibody has high affinity and high specificity, and can be used for OX40 detection and targeted treatment.

Description

Anti-OX40 single-domain antibodies and their applications Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, it relates to single-domain antibodies against OX40 and their applications. Background Technology

[0002] The interaction between OX40 and its ligand OX40L plays a crucial role in regulating immune responses.

[0003] On the one hand, activation of the OX40 / OX40L signaling pathway can enhance T cell survival, cytokine production, and the immune response of tumor-specific effector T cells, making OX40 a potential target for cancer therapy. In cancer treatment, OX40 agonists can promote CD4+ activation. + and CD8 + T cell survival enhances the production of tumor-specific memory T cells.

[0004] On the other hand, blocking the OX40 / OX40L signaling pathway can reduce T cell survival and cytokine production, leading to the research on OX40 for the treatment of autoimmune diseases. Inhibiting the OX40 / OX40L pathway can reduce immune activity in autoimmune diseases, and clinical trials and animal models have demonstrated that blocking OX40 or OX40L can have a therapeutic effect on autoimmune diseases.

[0005] Therefore, OX40 is a target with significant potential in the treatment of cancer and autoimmune diseases. Developing single-domain antibodies targeting OX40 to activate OX40 or block the binding of its ligand OX40L is of great importance for the treatment of cancer and autoimmune diseases. Summary of the Invention

[0006] This invention provides a single-domain antibody targeting OX40 and its application.

[0007] In a first aspect of the invention, a single-domain antibody against OX40 is provided, said single-domain antibody having three complementarity-determining regions (CDRs) derived from the VHH chain shown in the following amino acid sequences: SEQ ID NO: 1-10;

[0008] The CDRs are CDR1, CDR2, and CDR3 determined by any one of the IMGT rule, Kabat rule, Chothia rule, AbM rule, or Contact rule.

[0009] In another preferred embodiment, CDR1, CDR2 and CDR3 are selected from the group consisting of:

[0010] (A) Selected from the following groups: CDR1, CDR2 and CDR3:

[0011] (A1) CDR1 with amino acid sequence as shown in SEQ ID NO:11, CDR2 with amino acid sequence as shown in SEQ ID NO:12, and CDR3 with amino acid sequence as shown in SEQ ID NO:13;

[0012] (A2) CDR1 with amino acid sequence as shown in SEQ ID NO:14, CDR2 with amino acid sequence as shown in SEQ ID NO:15, and CDR3 with amino acid sequence as shown in SEQ ID NO:16;

[0013] (A3) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:18, and CDR3 with amino acid sequence as shown in SEQ ID NO:16;

[0014] (A4) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:19, and CDR3 with amino acid sequence as shown in SEQ ID NO:16;

[0015] (A5) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:18, and CDR3 with amino acid sequence as shown in SEQ ID NO:13;

[0016] (A6) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:20, and CDR3 with amino acid sequence as shown in SEQ ID NO:21;

[0017] (A7) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:22, and CDR3 with amino acid sequence as shown in SEQ ID NO:13;

[0018] (A8) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:20, and CDR3 with amino acid sequence as shown in SEQ ID NO:23;

[0019] (B) Selected from the following groups: CDR1, CDR2 and CDR3:

[0020] (B1) CDR1 with amino acid sequence as shown in SEQ ID NO:24, CDR2 with amino acid sequence as shown in SEQ ID NO:25, and CDR3 with amino acid sequence as shown in SEQ ID NO:26;

[0021] (B2) CDR1 with amino acid sequence as shown in SEQ ID NO:27, CDR2 with amino acid sequence as shown in SEQ ID NO:28, and CDR3 with amino acid sequence as shown in SEQ ID NO:29.

[0022] In another preferred embodiment, the amino acid sequence of the single-domain antibody includes any of the sequences shown in SEQ ID NO:1 to 10.

[0023] In another preferred embodiment, the amino acid sequence of the single-domain antibody includes any of the sequences shown in SEQ ID NO:1 to 8.

[0024] In another preferred embodiment, the amino acid sequence of the single-domain antibody includes any of the sequences shown in SEQ ID NO: 9 to 10.

[0025] In another preferred embodiment, the amino acid sequence of the single-domain antibody includes any of the sequences shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0026] In another preferred embodiment, the single-domain antibody is a humanized antibody comprising the sequence shown in any one of SEQ ID NO:30 to 39.

[0027] In another preferred embodiment, the single-domain antibody is a humanized antibody comprising the sequence shown in any one of SEQ ID NO:40 to 45.

[0028] In another preferred embodiment, the single-domain antibody is a humanized antibody comprising the sequence shown in any one of SEQ ID NO:46 to 50.

[0029] In another preferred embodiment, the CDR region of the single-domain antibody VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0030] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derivative sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining OX40 binding affinity.

[0031] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, more preferably 1-2, and even more preferably 1.

[0032] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0033] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0034] In another preferred embodiment, the frame region FR is of human, mouse, rabbit, or camel origin.

[0035] In another preferred embodiment, the nanobody is bound to human, mouse, or monkey-derived OX40.

[0036] In another preferred embodiment, the antibody is a heavy chain antibody, which includes heavy chain constant regions CH2 and CH3 (Fc segment).

[0037] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0038] In another preferred embodiment, the VHH chain of the single-domain antibody targeting OX40 has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the amino acid sequences shown in SEQ ID NO:1-10.

[0039] In another preferred embodiment, the VHH chain of the anti-OX40 single-domain antibody has one or more amino acid sequences as shown in SEQ ID NO:1-10.

[0040] In another preferred embodiment, the anti-OX40 single-domain antibody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0041] In another preferred embodiment, the amino acid sequence of the VHH chain of the single-domain antibody is selected from the group consisting of any one of the sequences shown in SEQ ID NO: 1 to 10.

[0042] In a second aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0043] (i) the single-domain antibody described in the first aspect of the present invention; and

[0044] (ii) Optional tag sequences to assist in expression and / or purification.

[0045] In another preferred embodiment, the tag sequence includes Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, 6His tag, or a combination thereof.

[0046] In another preferred embodiment, the recombinant protein specifically binds to OX40.

[0047] In another preferred embodiment, the recombinant protein includes a fusion protein.

[0048] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0049] In another preferred embodiment, the tag sequence is an Fc tag.

[0050] In another preferred embodiment, the recombinant protein comprises a fusion protein.

[0051] In another preferred embodiment, the recombinant protein is a fusion protein, and the fusion protein has a structure from the N-terminus to the C-terminus as shown in Formula I:

[0052] Z1-L-Z2 (Formula I)

[0053] In the formula,

[0054] Z1 is the VHH chain of the anti-OX40 single-domain antibody as described in the first aspect of the present invention;

[0055] L represents the connector sequence;

[0056] Z2 is the Fc segment of an immunoglobulin.

[0057] In a third aspect of the invention, a chimeric antigen receptor (CAR) is provided, the CAR containing an extracellular domain comprising the VHH chain of the single-domain antibody described in the first aspect of the invention.

[0058] In another preferred embodiment, the CAR has the structure shown in Formula Ia:

[0059] L-Nb-H-TM-C-CD3ζ (Ia)

[0060] In the formula,

[0061] L represents the absence of a signal peptide sequence;

[0062] Nb is a specific binding domain targeting OX40, which comprises the VHH chain of a single-domain antibody as described in the first aspect of the present invention.

[0063] H represents the area with no hinge or no connection.

[0064] TM represents a transmembrane domain;

[0065] C is the co-stimulation signal structure domain;

[0066] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers);

[0067] The "-" connects the peptide or peptide bond.

[0068] In another preferred embodiment, L is a signal peptide selected from the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or combinations thereof.

[0069] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0070] In another preferred embodiment, the TM is selected from the transmembrane regions of the following histones: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants / modified forms thereof, or combinations thereof.

[0071] In another preferred embodiment, C is selected from the co-stimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0072] In a fourth aspect of the invention, a polynucleotide is provided that encodes the single-domain antibody described in the first aspect of the invention.

[0073] In another preferred embodiment, the polynucleotide includes DNA, RNA, or cDNA.

[0074] In a fifth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide described in the fourth aspect of the invention.

[0075] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector includes viral vectors such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0076] In another preferred embodiment, the expression vector further includes a selection from the group consisting of promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), etc.

[0077] In a sixth aspect of the invention, a host cell is provided, the host cell containing the vector described in the fifth aspect of the invention, or having the polynucleotide described in the third aspect of the invention integrated into its genome.

[0078] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0079] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0080] In a seventh aspect of the invention, an engineered immune cell is provided, said engineered immune cell expressing a chimeric antigen receptor as described in a third aspect of the invention.

[0081] In another preferred embodiment, the engineered immune cells are selected from the group consisting of:

[0082] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0083] (ii) Chimeric antigen receptor γδ T cells (CAR-T cells);

[0084] (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells);

[0085] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0086] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or combinations thereof.

[0087] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0088] In an eighth aspect of the present invention, a method for generating an OX40 single-domain antibody is provided, comprising the steps of:

[0089] (a) The host cells described in the sixth aspect of the invention are cultured under conditions suitable for generating single-domain antibodies to obtain a culture containing the anti-OX40 single-domain antibody; and

[0090] (b) Isolate or recover the anti-OX40 single-domain antibody from the culture.

[0091] In another preferred embodiment, the anti-OX40 single-domain antibody has an amino acid sequence as shown in any of SEQ ID NO:1 to 10.

[0092] In a ninth aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0093] (a) The anti-OX40 single-domain antibody according to the first aspect of the present invention; and

[0094] (b) A conjugation portion conjugated to the single-domain antibody, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0095] In another preferred embodiment, the single-domain antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0096] In another preferred embodiment, the coupling portion is a drug or toxin.

[0097] In another preferred embodiment, the drug is a cytotoxic drug.

[0098] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0099] In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubule inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0100] In another preferred embodiment, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methamphetamine, pyrine, pyrine A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxychloroquine. Anthraxone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0101] In another preferred embodiment, the coupling portion is a detectable marker.

[0102] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2), antisense oligonucleotides, small interfering RNA, microRNAs, nucleic acid aptamers, antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanoran, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0103] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) anti-OX40 single-domain antibody as described in the first aspect of the invention. The multivalent meaning is that the amino acid sequence of the immunoconjugate contains a plurality of repeating anti-OX40 single-domain antibodies as described in the first aspect of the invention.

[0104] In another preferred embodiment, the term "multivalent" means that the amino acid sequence of the immunoconjugate contains a plurality of repeating anti-OX40 single-domain antibodies as described in the first aspect of the invention.

[0105] In a tenth aspect of the invention, the use of a single-domain antibody as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, an engineered immune cell as described in the seventh aspect of the invention, or an immunoconjugate as described in the ninth aspect of the invention, for the preparation of pharmaceuticals, reagents, detection plates, or kits is provided.

[0106] The reagents, detection plates, or kits are used to detect OX40 protein in samples.

[0107] The drug is used to prevent and / or treat diseases associated with abnormal OX40 expression or function.

[0108] In another preferred embodiment, the diseases associated with abnormal OX40 expression or function are selected from the group consisting of cancer and autoimmune diseases.

[0109] In another preferred embodiment, the cancer includes cancers selected from the group consisting of: breast cancer, melanoma, B-cell lymphoma, head and neck cancer, colon cancer, stomach cancer, prostate cancer, renal cell carcinoma, lung cancer, uterine carcinosarcoma, urothelial carcinoma, nasopharyngeal carcinoma, triple-negative breast cancer, or combinations thereof.

[0110] In another preferred embodiment, the autoimmune disease includes diseases selected from the group consisting of: moderate atopic dermatitis, severe atopic dermatitis, systemic lupus erythematosus, asthma, alopecia areata, or a combination thereof.

[0111] In an eleventh aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0112] (i) the single-domain antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the engineered immune cells described in the seventh aspect of the present invention, or the immunoconjugate described in the ninth aspect of the present invention; and

[0113] (ii) Pharmaceutically acceptable carriers.

[0114] In another preferred embodiment, the carrier is an excipient or a diluent.

[0115] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0116] In a twelfth aspect of the present invention, an OX40 protein detection reagent is provided, the detection reagent comprising:

[0117] (i) the anti-OX40 single-domain antibody as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the engineered immune cells as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention; and

[0118] (ii) A detectable carrier.

[0119] In another preferred embodiment, the coupling portion of the immunoconjugate is a diagnostic isotope.

[0120] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0121] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0122] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0123] In another preferred embodiment, the test reagent is in liquid or powder form (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, or inhaler).

[0124] In a thirteenth aspect of the present invention, a kit is provided, the kit comprising:

[0125] (1) A first container containing the single-domain antibody as described in the first aspect of the present invention; and / or

[0126] (2) A second container containing a secondary antibody against the single-domain antibody of the present invention;

[0127] or,

[0128] The kit contains the detection reagents as described in the twelfth aspect of the present invention.

[0129] In a fourteenth aspect of the present invention, a method for detecting OX40 protein in a sample is provided, the method comprising the steps of:

[0130] (1) Contact the sample with the single-domain antibody described in the first aspect of the present invention;

[0131] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of OX40 protein in the sample.

[0132] In another preferred embodiment, the method is an in vitro method.

[0133] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0134] In a fifteenth aspect of the invention, a method for treating a disease associated with abnormal OX40 expression or function is provided, comprising: administering to a subject in need an effective amount of a single-domain antibody as described in a first aspect of the invention, or a recombinant protein as described in a second aspect of the invention, engineered immune cells as described in a seventh aspect of the invention, an immunoconjugate as described in a ninth aspect of the invention, or a pharmaceutical composition as described in an eleventh aspect of the invention.

[0135] In another preferred embodiment, the object includes mammals, such as humans.

[0136] In another preferred embodiment, the diseases associated with abnormal OX40 expression or function are selected from the group consisting of cancer and autoimmune diseases.

[0137] In another preferred embodiment, the cancer includes cancers selected from the group consisting of:

[0138] Breast cancer, melanoma, B-cell lymphoma, head and neck cancer, colon cancer, stomach cancer, prostate cancer, renal cell carcinoma, lung cancer, uterine carcinosarcoma, urothelial carcinoma, nasopharyngeal carcinoma, triple-negative breast cancer, or combinations thereof.

[0139] In another preferred embodiment, the autoimmune disease includes diseases selected from the group consisting of: moderate atopic dermatitis, severe atopic dermatitis, systemic lupus erythematosus, asthma, alopecia areata, or a combination thereof.

[0140] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0141] Figure 1 shows the ELISA results for the VHH pool of human OX40-his.

[0142] Figure 2 shows the binding ability of anti-OX40 single-domain antibody to human OX40 antigen as detected by ELISA.

[0143] Figure 3 shows the binding ability of the anti-OX40 single-domain antibody to the rhesus monkey OX40 antigen as detected by ELISA.

[0144] Figure 4 shows the ability of anti-OX40 single-domain antibodies to block or promote the binding of ligand OX40L to antigen as detected by ELISA. Left: Candidate antibody promotes the binding of ligand OX40L to antigen; Right: Candidate antibody blocks the binding of ligand OX40L to antigen.

[0145] Figure 5 shows the binding ability of the anti-OX40 single-domain antibody to CHO-K1 cells that highly express hOX40, as detected by flow cytometry.

[0146] Figure 6 shows the activation of the hOX40 reporter cell line by Nb021-A002.

[0147] Figure 7 shows the synergistic activation of Nb021-A002 and ligand OX40L as analyzed by GraphPad Prism.

[0148] Figure 8 shows the synergistic activation effect of Nb021-A002 and ligand OX40L as analyzed by CompuSyn.

[0149] Figure 9 shows the Nb021-A253 blocking ligand OX40L activation activity.

[0150] Figure 10 shows the weight changes in a mouse model of atopic dermatitis during drug administration.

[0151] Figure 11 shows the changes in ear thickness in a mouse model of atopic dermatitis during drug administration.

[0152] Figure 12 shows the changes in redness and swelling scores in a mouse model of atopic dermatitis during drug administration.

[0153] Figure 13 shows the changes in desquamation scores during drug administration in a mouse model of atopic dermatitis.

[0154] Figure 14 shows the changes in clinical scores during the administration of the atopic dermatitis mouse model.

[0155] Figure 15 shows the rate of weight change in the monkey model of atopic dermatitis during the administration period.

[0156] Figure 16 shows the pharmacokinetic analysis of A253-h118 in a monkey model of atopic dermatitis.

[0157] Figure 17 shows the changes in ear thickness in a monkey model of atopic dermatitis during drug administration.

[0158] Figure 18 shows the changes in clinical scores during the administration of the atopic dermatitis monkey model.

[0159] Figure 19 shows the number of scratches in the monkey model of atopic dermatitis during the drug administration period.

[0160] Figure 20 shows the endpoint pathological scores of the monkey model of atopic dermatitis.

[0161] Figure 21 shows the endpoint cytokine analysis of the lesion site in a monkey model of atopic dermatitis.

[0162] Figure 22 shows the binding ability of the humanized Nb021-A002 single-domain antibody to CHO-K1 cells with high hOX40 expression as detected by flow cytometry.

[0163] Figure 23 shows the binding ability of the humanized Nb021-A253 single-domain antibody to HPB-ALL cells with high hOX40 expression as detected by flow cytometry.

[0164] Figure 24 shows the binding ability of the Nb021-A253 affinity maturation single-domain antibody to HPB-ALL cells with high hOX40 expression, as detected by flow cytometry. Detailed Implementation

[0165] Through extensive and in-depth research and screening, the inventors have, for the first time, developed a class of single-domain antibodies against OX40 and their humanized counterparts. Experimental results show that the single-domain antibodies and their humanized counterparts of this invention exhibit specific high affinity for OX40 and possess either OX40-activating activity or OX40 ligand-blocking activity. The single-domain antibodies of this invention with OX40-activating activity can activate OX40 alone or synergistically with the OX40 ligand OX40L; the single-domain antibodies with OX40 ligand-blocking activity exhibit a dose-response relationship. The single-domain antibodies and their humanized counterparts of this invention can specifically bind to human OX40 protein (including OX40 on the cell surface) and also possess human-monkey cross-binding activity. Based on these findings, this invention was completed.

[0166] the term

[0167] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0168] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0169] The term “giving” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0170] The term "EC50" refers to the concentration for 50% of the maximal effect, which is the concentration at which the maximal effect is achieved.

[0171] The term "IC50" refers to the half-inhibitory concentration of the antagonist being measured.

[0172] OX40

[0173] OX40, also known as TNFRSF4 (TNF receptor superfamily member 4) or CD134, is a receptor primarily found in activated CD4+ receptors. + and CD8 +OX40 is a co-stimulatory molecule expressed on T cells. Its ligand in vivo is OX40L (also known as CD152 or TNFSF4), which exists in a trimer form. OX40 / OX40L signaling plays a crucial role in T cell activation and proliferation. Studies have shown that OX40 antibodies can effectively promote T cell activation and proliferation, block the immunosuppressive effects of Treg cells, and activate the immune system.

[0174] The nanobody of the present invention

[0175] As used herein, the terms "single-domain antibody targeting OX40 of the present invention," "single-domain antibody of the present invention," "OX40 single-domain antibody of the present invention," "nanobody of the present invention," and "anti-human OX40 nanobody of the present invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to OX40 (including human OX40). Particularly preferred are single-domain antibodies with VHH chain amino acid sequences as shown in SEQ ID NO:1-10.

[0176] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0177] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning: to clone the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies that are naturally missing the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0178] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0179] As those skilled in the art will know, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the described anti-OX40 antibody or fragments thereof.

[0180] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0181] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0182] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0183] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0184] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to OX40 proteins, such as proteins or peptides having a heavy chain variable region. They may or may not contain initiating methionine.

[0185] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0186] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0187] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0188] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0189] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0190] The antibody of this invention refers to a polypeptide having OX40 protein-binding activity and including the aforementioned CDR region. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibody of this invention.

[0191] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0192] The present invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the nanobodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0193] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0194] Table 1

[0195] Polynucleotides, vectors and host cells

[0196] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0197] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0198] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0199] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0200] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0201] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0202] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0203] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0204] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0205] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0206] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0207] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0208] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0209] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0210] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0211] Immunoconjugates

[0212] The present invention also provides immunoconjugates (ADCs) based on the antibodies of the present invention, preferably nanobody-drug conjugates (NDCs).

[0213] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of the following: a toxic protein, a chemotherapeutic agent, a small molecule drug, an agonist small molecule (STING, TLR7, TLR8, etc.), or a radionuclide.

[0214] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0215] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0216] Single-domain antibodies can be conjugated to drugs to form antibody-drug conjugates (NDCs). Typically, an NDC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0217] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0218] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0219] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0220] The immunoconjugated drug of the present invention can also be a radionuclide conjugated drug (RDC), which is composed of the antibody of the present invention conjugated with a radionuclide.

[0221] In this invention, the drug-linker can be used to form NDC in a simple step. In other embodiments, bifunctional linker compounds can be used to form NDC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form NDC.

[0222] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0223] The present invention also provides a method for preparing NDC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (NDC).

[0224] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0225] In some embodiments, the structure of the immunoconjugate, preferably a single-domain antibody-drug conjugate NDC, is shown in the following molecular formula:

[0226] in:

[0227] nAb refers to the aforementioned single-domain antibody targeting OX40, heavy-chain antibody targeting OX40, or multispecific antibody.

[0228] LU stands for connector / connector;

[0229] D is a drug;

[0230] And the subscript p is a value selected from 1 to 10.

[0231] Pharmaceutical Composition

[0232] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0233] The pharmaceutical compositions of the present invention can be directly used to bind to OX40 protein molecules, and therefore can be used to treat allergies. Furthermore, other therapeutic agents can be used simultaneously.

[0234] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described nanobody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0235] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0236] Labeled single-domain antibodies

[0237] In a preferred embodiment of the invention, the nanobody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0238] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, a single-domain antibody against OX40 is labeled with colloidal gold to obtain colloidal gold-labeled nanobodies.

[0239] The anti-human OX40 single-domain antibody of the present invention has excellent specificity and high potency.

[0240] Phage display technology

[0241] The principle of phage display technology is to insert a foreign gene into an appropriate position in the structural gene of the phage coat protein. When the reading frame is normal and the normal function of the coat protein is not affected, the foreign gene will be expressed along with the expression of the coat protein, thus displaying the polypeptide or protein as a fusion protein on the phage surface. The displayed protein can maintain a relatively independent spatial structure and biological activity, which is conducive to the binding of the target protein. Therefore, the target protein can be used for rapid screening of phage display antibody libraries.

[0242] After the display library is constructed, it is incubated with the target protein as a stationary phase for a period of time. Unbound phages are washed away, and then the adsorbed phages are eluted using a competitive receptor. The eluted phages infect the host bacteria to multiply and expand, and then the next round of elution is performed.

[0243] After 2 to 5 rounds of "adsorption-elution-amplification" (more rounds of elution are required for some antibodies with weak affinity), a high enrichment of phages that can specifically bind to the target protein can be obtained.

[0244] Detection methods

[0245] This invention also relates to a method for detecting OX40 protein. The method generally involves the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of OX40 protein in the dissolved samples.

[0246] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0247] Reagent test kit

[0248] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0249] This invention also provides a detection kit for detecting OX40 levels, comprising an antibody that recognizes the OX40 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0250] application

[0251] As described above, the nanobody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of OX40-related diseases, basic medical research, and biological research. A preferred application is for clinical diagnosis and targeted therapy against OX40.

[0252] In another preferred embodiment, the treatment includes treating a disease associated with abnormal OX40 expression or function. The disease is cancer or an autoimmune disease.

[0253] In another preferred embodiment, the cancer includes cancers selected from the group consisting of: breast cancer, melanoma, B-cell lymphoma, head and neck cancer, colon cancer, stomach cancer, prostate cancer, renal cell carcinoma, lung cancer, uterine carcinosarcoma, urothelial carcinoma, nasopharyngeal carcinoma, triple-negative breast cancer, or combinations thereof.

[0254] In another preferred embodiment, the autoimmune disease includes diseases selected from the group consisting of: moderate atopic dermatitis, severe atopic dermatitis, systemic lupus erythematosus, asthma, alopecia areata, or a combination thereof.

[0255] The main advantages of this invention include:

[0256] (a) The present invention provides humanized single-domain antibodies that reduce the immunogenicity of the antibodies and improve their in vivo safety.

[0257] (b) This invention provides a single-domain antibody with cross-binding activity between human and monkey species.

[0258] (c) The production of the single-domain antibody of the present invention is simple.

[0259] (d) The single-domain antibody of the present invention having OX40 activation activity can synergistically activate OX40 with ligand OX40L.

[0260] (e) The single-domain antibody of the present invention having OX40 ligand blocking activity can effectively block the activity of ligand OX40L binding to OX40.

[0261] (f) The ligand-blocking single-domain antibody of the present invention showed good efficacy in a mouse model of atopic dermatitis.

[0262] (g) The ligand-blocking single-domain antibody of the present invention showed good efficacy in a monkey model of atopic dermatitis.

[0263] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0264] Example 1: Production of anti-OX40 single-domain antibodies induced by immunization of alpacas and detection of serum titer

[0265] Healthy alpacas of appropriate age were selected. In the first and fifth weeks, 0.5 mg of human OX40-Fc antigen (ACRO, Cat#OX0-H5255) was administered via subcutaneous injection at multiple sites. In the third, fourth, seventh, and eighth weeks, 0.5 mg of human OX40-his antigen (SinoBiological, Cat#10481-H08H) was administered. Except for the initial immunization, which used complete Freund's adjuvant (CFA), subsequent immunizations used incomplete Freund's adjuvant (IFA). Starting from the sixth week, blood samples were collected weekly, and the serum titer of the target antibody was monitored.

[0266] Serum titer was determined by coating human OX40-his onto an ELISA plate and incubating overnight at 4°C; blocking at room temperature for 2 hours; adding 3-fold serially diluted serum and incubating at room temperature for 1 hour; adding secondary antibody and incubating for 1 hour; color development and termination; and measuring the OD450 value using an ELISA reader. The serum titer results are shown in Table 1. Serum titers at weeks 7 and 8 were both greater than 256K, meeting the requirements for library construction.

[0267] Table 1 - Serum titer detection

[0268] Example 2: Construction and screening of anti-OX40 single-domain antibody phage library

[0269] Peripheral blood mononuclear cells (PBMCs) were isolated from alpaca cells at weeks 7 and 8. Total RNA was extracted from immunized alpaca PBMCs and further reverse transcribed into cDNA to obtain all antibody nucleotide sequences. These antibody nucleotide sequences were then constructed into a phage display vector using molecular cloning technology. The constructed vector was then electroporated into E. coli to obtain an alpaca immune phage display library.

[0270] The library size of the single-domain antibody library was determined by dilution plasmography. The total size of the phage display library constructed using peripheral blood PBMCs from week 7 was found to be 4.5 x 10⁻⁶.8 The total volume of the phage display library constructed using peripheral blood PBMCs from week 8 was 3.8 x 10⁻⁶. 8 Eighty single clones were selected from each of the two libraries for sequencing, and the sequencing results were further analyzed. Based on the total library size, insertion rate, and diversity sequencing analysis results, the effective library sizes of the two anti-OX40 single-domain antibody phage libraries were determined to be 3.36 x 10⁻⁶. 8 and 3.8 x 10 8 .

[0271] Antibody screening employed a cross-phase and liquid-phase screening method, targeting the human OX40, His Tag, and human OX40, Fc Tag antigens to screen phage libraries containing the two anti-OX40 single-domain antibodies. The experimental methods are as follows:

[0272] (1) Phage preparation

[0273] The alpaca immune phage or the obtained output set is used to prepare enriched phages through a series of steps, including inoculation, assisted phage infection, phage amplification, phage precipitation and resuspension, and then put into the next round of screening.

[0274] (2) Liquid phase separation

[0275] Biotin-labeled antigens are combined with streptavidin-conjugated magnetic beads, then incubated with prepared bacteriophages, washed, and eluted. After two rounds of panning, specific monoclonal antibodies against the antigen are enriched.

[0276] (3) Solid-phase sea separation

[0277] The antigen was coated on the surface of an immunotube with high adsorption capacity. Then, the prepared phage was added to the immunotube for incubation, washing and elution. After two rounds of panning, specific monoclonal antibodies against the antigen were enriched.

[0278] (4) Preliminary selection and testing

[0279] The enrichment effect of the selected output set was detected by ELISA. The process included plate coating, blocking, incubation, addition of secondary antibody, color development, termination, and detection of OD values. A standard curve was generated using serially diluted known standards to quantify the single-domain antibody expression supernatant. The supernatant concentration was plotted on the x-axis, and the OD value on the y-axis, with negative values ​​as a reference, to evaluate the enrichment degree of the output set (Figure 1).

[0280] (5) Initial screening of monoclonal antibodies

[0281] The method involves coating with target antigens, followed by a series of steps including coating, blocking, incubation, adding secondary antibody, color development, termination, and detection of OD values. Positive clones are identified based on a specific background value and then sent for testing.

[0282] After initial screening by ELISA, a total of 1066 monoclonal antibodies were selected, and 654 positive clones that were bound to human OX40 and His Tag antigens were obtained. Sequence analysis revealed 10 molecules with unique sequences (Table 2), involving 5 CDR1, 7 CDR2 and 7 CDR3 sequences (Table 3).

[0283] Table 2 - Sequences of Human OX40 Antigen Binding Positive Single-Domain Antibodies

[0284] Table 3 - Human OX40 antigen-binding positive CDR sequences

[0285] Example 3: Construction of anti-OX40 single-domain antibody expression strain and protein preparation

[0286] (1) Yeast expression system

[0287] A 6*his tag was added to the N-terminus of the anti-OX40 single-domain antibody. The codons of the above gene sequence were optimized and constructed into the pPICZ alpha A plasmid, which was then linearized using PemI enzyme. 5 μL of the linearized plasmid was added to 100 μL of competent cells, and the plasmid carrying the target gene was transformed into X33 Pichia pastoris cells using an electroporator (Biorad, MicroPulser) with the following parameters: 1.5 kV, 4 ms. After electroporation, the Pichia pastoris cells were revitalized using a mixed medium (YPD:Sorbitol = 1:1). Then, 50 μL of Pichia pastoris cells were plated on YPD solid medium containing 200, 400, 600, and 800 μg / mL zeocin (Invitrogen, Cat#R25001), respectively. High-copy strains of the target gene were obtained through zeocin concentration gradient selection.

[0288] Single-clone screening was performed by culturing single-clone strains in BMGY medium (Sangon, Cat#B540130) at 30℃ and 250 rpm. After obtaining sufficient bacterial cells, the target single-domain antibody was induced to secrete and express in BMMY medium (Sangon, Cat#B540131) at 20℃ and 250 rpm. After 24 hours, 20 μL of supernatant was collected, and the expression level of each colony was analyzed by gel electrophoresis. The strain with the highest expression level was selected for preservation and protein expression production. The method for large-scale protein expression and purification is as follows: 400 μL of bacterial culture was inoculated into 200 mL of BMGY medium and cultured at 30℃ and 250 rpm for 3 days for enrichment. Then, 80 mL of BMMY medium was used to induce expression, with 1% methanol added every 24 hours. The target single-domain antibody was induced to secrete and express for 3 days at 20℃ and 250 rpm.

[0289] After induction, the supernatant was collected by centrifugation at 12,000 rpm for 15 minutes in a high-speed refrigerated centrifuge. The protein was then purified using nickel affinity chromatography (Cytiva, Cat#17092108). Before use, the nickel affinity chromatography column was equilibrated with binding buffer. The supernatant containing the target protein was then passed through the column, allowing the His-tagged protein to bind to nickel ions and remain on the column. Non-specifically bound proteins were then washed away with buffer containing 20 mM imidazole. Finally, the target protein was eluted with elution buffer containing 250 mM imidazole.

[0290] (2) Mammalian expression system

[0291] The C-terminus of the VHH sequence was fused with the Fc fragment of human IgG1. After codon optimization, the fusion sequence was constructed into the pcDNA3.4 vector. The fusion expression plasmid was transiently transfected into Expi CHO cells for expression for 7 days in Expi CHO culture medium. TM Expression medium (Thermo Fisher, Cat#A2910001), transfection kit: ExpiFectamine TM CHO Transfection Kit (Thermo Fisher, Cat#A29129). After the expression process was completed, the cells were centrifuged, and the supernatant was collected, filtered through a 0.22 μM filter membrane, and then processed with Protein A affinity packing material (Cytiva, MabSelect SuRe). TM Purify VHH-Fc.

[0292] Example 4: Binding activity analysis of anti-OX40 single-domain antibody with human and monkey recombinant OX40 protein

[0293] The binding activity of the candidate antibody to human OX40-his tag (SinoBiological, Cat#10481-H08H) and monkey OX40-his tag (SinoBiological, Cat#90846-C08H) was analyzed using ELISA. The experimental method is as follows:

[0294] 1) Plate coating: Dilute the antigen with 1×PBS to a concentration of 2μg / mL, add 30μL / well to a 96-well ELISA plate, and coat overnight at 4°C.

[0295] 2) Blocking: Wash the plate 3 times with PBST, add blocking buffer (5% PBSM) and block at room temperature for 2 hours.

[0296] 3) Incubation: Wash the plate, add 30 μL of sample diluted with 1% PBSM per well, and incubate at room temperature for 60 minutes.

[0297] 4) Secondary antibody incubation: Wash the plate 3 times with PBST, add secondary antibody goat anti-human IgG-Fc-HRP (Abcam, Cat#ab97225), and incubate at room temperature for 50 minutes.

[0298] 5) Color development: Wash the plate 3 times with PBST, and add 30 μL TMB to each well.

[0299] 6) Termination: Add 2M termination solution to terminate the reaction and simultaneously measure OD450.

[0300] The binding EC50 values ​​were calculated by fitting the 4-parameter equation of the S-curve using GraphPad Prism 10 software. The experimental results showed that all candidate antibodies in the VHH-Fc form had cross-binding activity between humans and monkeys. Except for Nb021-B242, which had weaker monkey antigen binding activity, the human and monkey antigen binding activities of the other candidate antibodies were comparable to those of the positive control BGB-A445 (Figure 2-3).

[0301] Example 5: The activity of anti-OX40 single-domain antibody in blocking or promoting the binding of ligand OX40L to OX40.

[0302] 5.1 The binding activity of the candidate antibody to ligands OX40L (SinoBiological, Cat#13127-H04H) and OX40 (SinoBiological, Cat#10481-H08H) was analyzed by ELISA.

[0303] The experimental method is as follows:

[0304] 1) Plate coating: Dilute the ligand with 1×PBS to a concentration of 4 μg / mL, add 30 μL / well to a 96-well ELISA plate, and coat overnight at 4°C.

[0305] 2) Blocking: Wash the plate 3 times with PBST, add blocking buffer (5% PBSM) and block at room temperature for 2 hours.

[0306] 3) Premix: Mix the serially diluted primary antibody with 4 μg / mL of antigen at a 1:1 volume ratio and incubate at room temperature for 60 minutes.

[0307] 4) Incubation: Wash the plate, add 30 μL of premixed sample per well, and incubate at room temperature for 60 minutes.

[0308] 5) Secondary antibody incubation: Wash the plate 3 times with PBST, add secondary antibody anti-6*his-HRP (Protientech, Cat#HRP-66005), and incubate at room temperature for 50 minutes.

[0309] 6) Color development: Wash the plate 3 times with PBST, and add 30 μL TMB to each well.

[0310] 7) Termination: Add 2M termination solution to terminate the reaction and simultaneously measure OD450.

[0311] 5.2 ELISA method was used to analyze the binding activity of candidate antibodies in promoting the binding of ligands OX40L and OX40.

[0312] The experimental method is as follows:

[0313] 1) Plate coating: Dilute the ligand with 1×PBS to a concentration of 4 μg / mL, add 30 μL / well to a 96-well ELISA plate, and coat overnight at 4°C.

[0314] 2) Blocking: Wash the plate 3 times with PBST, add blocking buffer (5% PBSM) and block at room temperature for 2 hours.

[0315] 3) Premixing: Mix the serially diluted primary antibody with 0.5 μg / mL antigen at a volume ratio of 1:1 and incubate at room temperature for 60 minutes.

[0316] 4) Incubation: Wash the plate, add 30 μL of premixed sample per well, and incubate at room temperature for 60 minutes.

[0317] 5) Secondary antibody incubation: Wash the plate 3 times with PBST, add secondary antibody anti-6*his-HRP (Protientech, Cat#HRP-66005), and incubate at room temperature for 50 minutes.

[0318] 6) Color development: Wash the plate 3 times with PBST, and add 30 μL TMB to each well.

[0319] 7) Termination: Add 2M termination solution to terminate the reaction and simultaneously measure OD450.

[0320] The S-curve was fitted with a 4-parameter equation using GraphPad Prism 10 software, and the combined EC50 value was calculated (Figure 4).

[0321] Experimental results show that Nb021-A002, Nb021-A077, Nb021-A089, Nb021-A147, Nb021-A165, and Nb021-A172 have the activity of promoting the binding of ligand OX40L to OX40; while Nb021-A253 and Nb021-B242 have the activity of blocking the binding of ligand OX40L to OX40.

[0322] Example 6: Binding activity analysis of anti-OX40 single-domain antibody with human OX40-overexpressing cells

[0323] hOX40 CHO-K1 cells were obtained by stable transfection of CHO-K1 cells with the pIRES-Neo3 vector expressing the human OX40 gene (NM_003327.4).

[0324] The experimental method for cell binding assay of VHH-Fc candidate antibodies is as follows:

[0325] (1) Cell plating: Transfer cells from culture flasks to centrifuge tubes, centrifuge to remove supernatant, resuspend in culture medium, count, and adjust cell density to 1×10⁻⁶. 6 cells / mL. Take a 96-well round-bottom plate and add 100 μL of cells to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 minutes. Discard the supernatant.

[0326] (2) Addition of candidate antibody dilution buffer: Dilute the antibody with FACS buffer containing 2% FBS to prepare eight concentration gradients of 150.0000 nM. Add 100 μL of antibody dilution buffer to each well of a 96-well cell plate using a 12-channel pipette. Mix well and incubate at 4°C for 1 h.

[0327] (3) Addition of secondary antibody: The PE-labeled anti-human IgG-Fc (Jackson, Cat#109-115-098) secondary antibody was diluted 1:200 with FACS buffer containing 2% FBS. The cell culture plate was centrifuged to remove the supernatant, and 100 μL of the secondary antibody dilution buffer was added to the cell culture plate using a 100 μL 12-channel pipette. The cell culture plate was incubated at 4°C for 30 minutes. After centrifugation to remove the supernatant, the plate was washed twice with FACS buffer, and the cells were resuspended in 120 μL of FACS buffer in each well.

[0328] (4) Data acquisition: Turn on the flow cytometer and after the instrument is cleaned, measure the average fluorescence intensity of each sample.

[0329] The experimental method for cell binding assay of VHH form candidate antibodies is as follows:

[0330] Cells in the logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS), and the cell density was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells / mL, add 180 μL / well of cell suspension to a 96-well U-shaped plate. Dilute the test sample stock solution with flow cytometry buffer to prepare serially diluted 10* concentration antibody solutions. Add 20 μL of the above solution to the cell suspension in the 96-well plate, vortex to mix, and incubate the 96-well plate at 4°C for 30 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, add 200 μL / well of 1:1000 diluted iFluor647-labeled rabbit anti-camel VHH antibody (Genscript, Cat#A02019) solution, vortex to mix, and incubate the 96-well plate at 4°C for 30 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, and resuspend the cells in 200 μL / well of flow cytometry buffer. Measure the average fluorescence intensity of each sample using a flow cytometer (BD, FACSCelesta).

[0331] The S-curve was fitted with a 4-parameter equation using GraphPad Prism 10 software, and the combined EC50 value was calculated (Figure 5).

[0332] Experimental results showed that all candidate antibodies could bind to human OX40 expressed on the cell surface.

[0333] Example 7: Analysis of OX40 receptor activation activity of Nb021-A002 single-domain antibody in reporter cell lines

[0334] Using CHO-K1 cells as the host cell line, hOX40 reporter cell lines (NF-κB-Luc2P / hOX40 CHO-K1) were constructed by overexpressing hOX40 and a luciferase reporter gene regulated by the NF-κB response element. Cells were seeded at a density of 30,000 cells / 90 μL / well in cell culture plates and incubated overnight at 37°C. Serially diluted 10-fold antibody solutions were prepared and added at 10 μL / well to each cell culture plate, creating an antibody treatment group (with antibody) and a zero-concentration control group (without antibody). Each concentration point was set up in triplicate. The cell culture plates were incubated at 37°C for another 6 hours. Cells were treated with a luciferase reporter gene assay kit (Novizan, Cat#DD1203), and the chemiluminescence intensity (RLU) of each well was measured using a microplate reader. The fold change (Fold Induction) was calculated by dividing the chemiluminescence intensity of the antibody-treated group by the chemiluminescence intensity of the zero-concentration control group. The EC50 value was then calculated by fitting the sigmoid curve with a 4-parameter equation using GraphPad Prism 10 software.

[0335] The experimental results showed that the Nb021-A002 single-domain antibody activated the hOX40 reporter cell line to produce a reporter signal, and there was a clear dose-response relationship between concentration and fold change. The negative control single-domain antibody GX-35-28 did not produce a reporter signal (Figure 6).

[0336] Example 8: Nb021-A002 single-domain antibody and ligand OX40L synergistically activate hOX40 receptor in reporter cell lines

[0337] hOX40 reporter cell lines were seeded into cell culture plates at a density of 30,000 cells / 80 μL / well and incubated overnight at 37°C. Serially diluted 10-fold antibody and hOX40L (Cat#OXL-HM140) solutions were prepared and added to cell culture plates at 10 μL / well, with triplet wells for each concentration point, serving as the single-drug treatment group. In the combined treatment group, 10 μL of each of the serially diluted 10-fold antibody and hOX40L solutions was added to cell culture plates, allowing the cells to be treated with a series of antibody concentrations and a fixed concentration of hOX40L (3.125 ng / mL).

[0338] The experimental groups were set up as follows: antibody-only group (serialized antibody concentrations, no ligand), ligand-only group (no antibody, serialized ligand concentrations), antibody and ligand combination group (serialized antibody concentrations, fixed ligand concentration), maximum activation control group (no antibody, 200 ng / mL ligand), and non-activation control group (no antibody, no ligand). Cell plates were incubated at 37°C for 6 hours. Cells were treated with a luciferase reporter gene assay kit (Novizan, Cat#DD1203), and the chemiluminescence intensity of each well was measured using a microplate reader. The fold change was calculated by dividing the chemiluminescence intensity of the single-drug group, combination group, and maximum activation control group by the chemiluminescence intensity of the non-activation control group. The EC50 value was calculated using a 4-parameter S-curve fitted with GraphPad Prism 10 software. The fold change in the monotherapy and combination therapy groups was divided by the fold change in the maximum activation control group to calculate the affected score (Fa). The combination index (CI) was analyzed using CompuSyn software.

[0339] The experimental results showed that the combination index of Nb021-A002 single-domain antibody and hOX40L was less than 1, indicating that the two have a synergistic effect in activating the hOX40 receptor (Figures 7 and 8).

[0340] Example 9: Ligand blocking activity analysis of Nb021-A253 single-domain antibody in reporter cell lines

[0341] hOX40 reporter cell lines were seeded into cell culture plates at a density of 30,000 cells / 80 μL / well and incubated overnight at 37°C. Serially diluted 10* antibody solutions were added to each well at 10 μL, with triplet wells for each concentration. The cell culture plates were pre-incubated at 37°C for 15 minutes. A 10-fold concentration of hOX40L solution was then added to each well, creating antibody treatment groups (antibody and ligand), ligand activation control groups (no antibody, ligand), and non-activation control groups (no antibody, no ligand). The cell culture plates were incubated at 37°C for 6 hours. Cells were treated with a luciferase reporter gene assay kit (Novizan, Cat#DD1203), and the chemiluminescence intensity of each well was measured using a microplate reader. The fold change was calculated by dividing the chemiluminescence intensity of the antibody-treated group or the ligand-activated control group by the chemiluminescence intensity of the non-activated control group. The EC50 value was then calculated by fitting the sigmoid curve with a 4-parameter equation using GraphPad Prism 10 software.

[0342] Experimental results showed that the Nb021-A253 single-domain antibody inhibited the activation activity of the ligand, thereby reducing the reporter signal, and there was a clear dose-response relationship between concentration and fold change (Figure 9).

[0343] Example 10: Nb021-A253 single-domain antibody showed significant efficacy in a mouse model of atopic dermatitis.

[0344] Nb021-A253 was tandemly linked with an anti-serum albumin single-domain antibody to increase the half-life of Nb021-A253 in mice. The tandem antibody was named A253-h118, and its sequence is as follows:

[0345] After optimizing the tandem antibody codons, the antibody was expressed using a yeast expression system and purified by nickel column affinity chromatography. Finally, the buffer for A253-h118 was replaced with PBS by gel filtration chromatography.

[0346] Animal experiments were conducted using OX40 / OX40L dual-target humanized mice. A mouse model of atopic dermatitis was induced by intradermal injection of mouse-dermal TSLP in the ear. Eight B-hOX40 / hOX40L mice were randomly divided into two groups according to ear thickness and body weight: Group G1 was the Vehicle control group (n=4), and Group G2 was the A253-h118 experimental group (n=4). The day of grouping was defined as Day 0.

[0347] The administration method was tail vein injection, twice a week, 25 mg / kg each time, for a total of 6 administrations. During the administration period, mice were clinically observed for indicators such as body weight, ear thickness, degree of redness and swelling, and desquamation to evaluate the efficacy of the test substance.

[0348] Summary of experimental results: (1) The mice gained weight steadily without any obvious abnormalities, indicating that the drug was safe at this dose (Figure 10). (2) Compared with the Vehicle control group, the mice in the A253-h118 experimental group had significantly reduced ear thickness, significantly improved ear redness and swelling and desquamation, and significantly reduced total clinical score (Figures 11-14), indicating that A253-h118 showed significant efficacy in the atopic dermatitis mouse model.

[0349] Example 11: A253-h118 showed significant efficacy in a monkey model of atopic dermatitis.

[0350] The preparation method for sample A253-h118 is the same as in Example 10.

[0351] Animal experiments used a DNCB-induced atopic dermatitis model in cynomolgus monkeys, and the specific procedures were as follows:

[0352] Six male cynomolgus macaques were used, with three macaques in each group, for a total of two groups. All animals underwent DNCB treatment on the shaved areas of their backs and ears to induce an atopic dermatitis model. Group G1 received the solvent subcutaneously; Group G2 received A253-h118 subcutaneously at a dose of 10 mg / kg; Group G3 received Amlitelimab subcutaneously at a dose of 10 mg / kg; and Group G4 received Rocatinlimab subcutaneously at a dose of 10 mg / kg. Dosing was administered once weekly for a total of four times. During the experiment, clinical observation, clinical scores, pruritus statistics, and skin samples were collected at the experimental endpoint for efficacy evaluation. Blood samples were also collected at different time points for pharmacokinetic studies.

[0353] Summary of experimental results: (1) During the experiment, apart from the appearance of atopic dermatitis-like symptoms at the induction site, no obvious abnormalities were observed in the clinical observation of the animals in each group. The weight fluctuation of all animals was relatively small, ranging from -5.26% to +10.71%, without significant decrease. This indicates that the drug has good safety at this dose (Figure 15). (2) A253-h118 has similar pharmacokinetic data to IgG antibodies Amlitelimab and Rocatinlimab (Figure 16), indicating that A253-h118 has a good half-life in monkeys. (3) Compared with the Vehicle control group, the ear thickness, clinical score, scratching frequency, and pathological score at the experimental endpoint of the mice in the A253-h118 experimental group were significantly reduced (Figures 17-20), indicating that A253-h118 showed significant efficacy in the DNCB-induced atopic dermatitis monkey model, and its efficacy was close to that of Amlitelimab and better than that of Rocatinlimab. (4) Analysis of cytokines in pathological tissues revealed that, compared with the Vehicle control group, A253-h118 could effectively downregulate IFN-γ, IL-17, IL-31 and IL-22 cytokines in the lesion site, and its downregulation ability was similar to that of Amlitelimab and better than that of Rocatinlimab (Figure 21).

[0354] Example 12: Humanization of Nb021-A002 and Nb021-A253 single-domain antibodies

[0355] Humanization of Nb021-A002 and Nb021-A253 was achieved by comparing the parental sequence with the human Germline database, defining the parental antibody CDR and framework region, and designing sequences with different degrees of humanization based on the differential sites in the framework region (Table 4).

[0356] Table 4 - Summary Table of Humanization Sequences for Nb021-A002

[0357] Example 13: Stability analysis of humanized Nb021-A002 and Nb021-A253 single-domain antibodies

[0358] The C-terminus of the humanized VHH sequence was fused with a human 6*his tag or flag tag. After codon optimization, the fusion sequence was constructed into the pcDNA3.4 vector. The fusion expression plasmid was transiently transfected into Expi CHO cells for expression for 7 days, and VHH was purified by affinity chromatography.

[0359] The aggregate formation tendency of humanized VHH was analyzed by HPLC-SEC. The chromatographic column used in the experiment was an XBridge BEH. SEC 3.5μm, 7.8×300mm (Waters, 186007640), flow rate set at 0.8mL / min; detection wavelength at 280nm.

[0360] The test results are shown in Table 5. The results indicate that the proportion of all humanized antibody monomers is higher than 98%, the samples are relatively stable, and they are not prone to forming aggregates.

[0361] Table 5 - Summary of SEC-HPLC Results for Humanized Nb021-A002

[0362] Note: " / " represents none.

[0363] The thermal stability of humanized VHH was further analyzed using differential scanning fluorescence (DSF).

[0364] The instrument used in the experiment was an ABI 7500 Fast Real-Time PCR instrument. The experiment type was selected as melting curve, and continuous mode was adopted. The scanning temperature was 25℃~99℃. The temperature corresponding to the first peak and trough of the derivative function of the melting curve was determined as the denaturation temperature Tm1 of the protein, the temperature corresponding to the second peak and trough was determined as the denaturation temperature Tm2 of the protein, and the temperature corresponding to the third peak and trough was determined as the denaturation temperature Tm3 of the protein. The detection results are shown in Table 6.

[0365] The results showed that, except for VHH2, which had a slightly higher Tm1 than the parent VHH, the Tm1 of all other humanized VHH molecules in Nb021-A002 was reduced to varying degrees. In contrast, the Tm1 of all humanized Nb021-A253 molecules was higher than that of the parent VHH.

[0366] Table 6 - Summary of DSF Results for Humanized Nb021-A002 and Nb021-A253

[0367] Example 14: Analysis of human OX40 protein binding activity of humanized Nb021-A002 single-domain antibody

[0368] The binding activity of humanized VHH to human OX40 protein (ACRO, Cat#OX0-H5255) was analyzed by surface plasmon resonance (SPR).

[0369] The instrument used was a biacore T200 (Cytiva). The test results are shown in Table 7. The affinity test results showed that all humanized VHHs bound human OX40 protein, and the binding activity was comparable to that of the parent.

[0370] Table 7 - Summary of SPR Results for Humanized Nb021-A002

[0371] Example 15: Cell binding activity analysis of humanized Nb021-A002 single-domain antibody

[0372] The cell binding assay used hOX40 CHO-K1 cells as target cells, and the C-terminus of the humanized VHH was tagged with a flag.

[0373] The experimental method for VHH-flag-form humanized antibody cell binding assay is as follows:

[0374] (1) Cell plating: Transfer cells from culture flasks to centrifuge tubes, centrifuge to remove supernatant, resuspend in culture medium, count, and adjust cell density to 1×10⁻⁶. 6 cells / mL. Take a 96-well round-bottom plate and add 100 μL of cells to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 minutes. Discard the supernatant.

[0375] (2) Addition of candidate antibody dilution buffer: Dilute the antibody with FACS buffer containing 2% FBS to prepare eight concentration gradients of 40 μg / ml. Add 100 μL of antibody dilution buffer to each well of a 96-well cell plate using a 12-channel 100 μL pipette. After mixing, incubate at 4°C for 1 h.

[0376] (3) Addition of secondary antibody: The PE-labeled anti-DYKDDDK tag antibody (Biolegend, Cat#637310) was diluted 1:100 with FACS buffer containing 2% FBS. The cell culture plate was centrifuged to remove the supernatant. 100 μL of the secondary antibody dilution buffer was added to the cell culture plate using a 100 μL 12-channel pipette. The cell culture plate was incubated at 4°C for 30 minutes. After centrifugation to remove the supernatant, the plate was washed twice with FACS buffer, and the cells were resuspended in 120 μL of FACS buffer in each well.

[0377] (4) Data acquisition: Turn on the flow cytometer and after the instrument is cleaned, measure the average fluorescence intensity of each sample.

[0378] The binding EC50 values ​​were calculated using sigmoid curves and 4-parameter equations fitted with GraphPadPrism10 software (Figure 22). The experimental results showed that all humanized antibodies could bind to human OX40 expressed on the cell surface, with VHH2 exhibiting the best cell-binding activity.

[0379] Example 16: Cell binding activity analysis of humanized Nb021-A253 single-domain antibody

[0380] The cell binding assay used HPB-ALL cells with high hOX40 expression as target cells, and the nanobody had an Fc tag at its C-terminus.

[0381] HPB-ALL cells in logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS), and the cell density was adjusted to 1 × 10⁻⁶ cells / year. 6 Cells / mL, add 180 μL / well of cell suspension to a 96-well U-shaped plate. Dilute the test sample stock solution with flow cytometry buffer to prepare serially diluted 10* concentration antibody solutions. Add 20 μL of the above solution to the cell suspension in the 96-well plate, vortex to mix, and incubate the 96-well plate at 4°C for 30 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, add 200 μL / well of 1:1000 diluted Alexa Fluor 647-labeled goat anti-human IgG (H+L) antibody (Yisheng Bio, Cat#33223ES60) solution, vortex to mix, and incubate the 96-well plate at 4°C for 30 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, and resuspend the cells in 200 μL / well of flow cytometry buffer. Measure the average fluorescence intensity of each sample using a flow cytometer (BD, FACSCelesta). The S-curve was fitted with a 4-parameter equation using GraphPad Prism 10 software, and the combined EC50 value was calculated.

[0382] Experimental results showed that all humanized VHH-Fc cells bound to HPB-ALL cells (Figure 23).

[0383] Example 17: Affinity maturation of humanized Nb021-A253 single-domain antibody

[0384] To enhance the affinity of humanized Nb021-A253, the inventors used the humanized antibody Nb021-hA253-VHH1 as the affinity maturation template and modified the antibody for affinity maturation. Affinity maturation employed single-point and double-point saturation mutagenesis methods, with primers designed specifically for the CDR region to construct an affinity maturation library. Phage display technology was used to screen the matured molecules using solid-phase, liquid-phase, and cell-based methods. After initial screening, 55 unique sequences were obtained, and expression and purification vectors were constructed from these sequences. ELISA, FACS, and SPR were then used for binding affinity analysis, ultimately yielding five antibodies with significantly higher affinity than the original template (Table 8).

[0385] Table 8 - Affinity-Maturing Single-Domain Antibody Sequences

[0386] Cell binding verification showed that the cell binding levels of the five matured antibodies were significantly higher than those of the humanized parent Nb021-hA253-VHH1 (3-14 times higher) and the original Nb021-A253 sequence (2-7 times higher). Cell binding data are shown in Figure 24. The experimental method was the same as in Example 16.

[0387] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

A single-domain antibody against OX40, characterized in that, The single-domain antibody has three complementarity-determining regions (CDRs) derived from the VHH chain shown in the following amino acid sequences: SEQ ID NO: 1-10; The CDRs are CDR1, CDR2, and CDR3 determined by any one of the IMGT rule, Kabat rule, Chothia rule, AbM rule, or Contact rule. The single-domain antibody as described in claim 1, characterized in that, CDR1, CDR2 and CDR3 are selected from the following group: (A) Selected from the following groups: CDR1, CDR2 and CDR3: (A1) CDR1 with amino acid sequence as shown in SEQ ID NO:11, CDR2 with amino acid sequence as shown in SEQ ID NO:12, and CDR3 with amino acid sequence as shown in SEQ ID NO:13; (A2) CDR1 with amino acid sequence as shown in SEQ ID NO:14, CDR2 with amino acid sequence as shown in SEQ ID NO:15, and CDR3 with amino acid sequence as shown in SEQ ID NO:16; (A3) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:18, and CDR3 with amino acid sequence as shown in SEQ ID NO:16; (A4) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:19, and CDR3 with amino acid sequence as shown in SEQ ID NO:16; (A5) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:18, and CDR3 with amino acid sequence as shown in SEQ ID NO:13; (A6) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:20, and CDR3 with amino acid sequence as shown in SEQ ID NO:21; (A7) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:22, and CDR3 with amino acid sequence as shown in SEQ ID NO:13; (A8) CDR1 with amino acid sequence as shown in SEQ ID NO:17, CDR2 with amino acid sequence as shown in SEQ ID NO:20, and CDR3 with amino acid sequence as shown in SEQ ID NO:23; (B) Selected from the following groups: CDR1, CDR2 and CDR3: (B1) CDR1 with amino acid sequence as shown in SEQ ID NO:24, CDR2 with amino acid sequence as shown in SEQ ID NO:25, and CDR3 with amino acid sequence as shown in SEQ ID NO:26; (B2) CDR1 with amino acid sequence as shown in SEQ ID NO:27, CDR2 with amino acid sequence as shown in SEQ ID NO:28, and CDR3 with amino acid sequence as shown in SEQ ID NO:

29. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the single-domain antibody of claim 1; and (ii) Optional tag sequences to assist in expression and / or purification. A polynucleotide, characterized in that, The polynucleotide encodes the single-domain antibody of claim 1. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 4. A host cell, characterized in that, The host cell contains the vector of claim 5, or the genome is integrated with the polynucleotide of claim 3. A method for generating OX40 single-domain antibodies, characterized in that, Including the following steps: (a) Culturing the host cells of claim 6 under conditions suitable for generating single-domain antibodies to obtain a culture containing the anti-OX40 single-domain antibody; and (b) Isolate or recover the anti-OX40 single-domain antibody from the culture. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) the anti-OX40 single-domain antibody according to claim 1; and (b) A conjugation portion conjugated to the single-domain antibody, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof. The use of the single-domain antibody as claimed in claim 1, the recombinant protein as claimed in claim 3, or the immunoconjugate as claimed in claim 8, is characterized in that, Used in the preparation of pharmaceuticals, reagents, test plates, or kits; The reagents, detection plates, or kits are used to detect OX40 protein in samples. The drug is used to prevent and / or treat diseases associated with abnormal OX40 expression or function. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the single-domain antibody of claim 1, the recombinant protein of claim 3, or the immunoconjugate of claim 8; and (ii) Pharmaceutically acceptable carriers.

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