Single-domain antibody for resisting 4-1BB and application of single-domain antibody

Abstract

The invention provides a single-domain antibody for resisting 4-1BB and application of the single-domain antibody. In particular, the invention provides a specific single domain antibody for resisting human 4-1BB. The invention also discloses a coding sequence for coding the single-domain antibody or the VHH chain thereof, a corresponding expression vector and a host cell, and a method for producing the single-domain antibody. The single-domain antibody provided by the invention has high affinity and high specificity, and can be used for detection and targeted therapy of 4-1BB.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a single-domain antibody against 4-1BB and its applications. Background Technology

[0002] The tumor necrosis factor receptor superfamily (TNFRSF) plays a crucial role in many biological events in organisms. This superfamily contains 29 members, which can be divided into three major classes based on their cytoplasmic sequences and signal transduction characteristics. Among them, 4-1BB, OX40, and GITR belong to the second class.

[0003] 4-1BB, also known as CD137 / TNFRSF9, is a type I transmembrane protein composed of an extracellular, transmembrane, and intracellular region. It is primarily expressed on the surface of activated T cells and is a T cell co-stimulatory molecule; its binding to the ligand 4-1BBL stimulates T cell activation and proliferation. Anti-4-1BB monoclonal antibodies selectively regulate CD8... + The proliferation of T cells and the expression of the pro-inflammatory cytokine IFNγ promote tumor clearance and have anti-tumor activity.

[0004] Single-domain antibodies are a novel type of antibody molecule discovered in camels by the research group of Belgian immunologists Hamers-Casterman. Compared to traditional antibodies, they offer advantages such as small molecular weight, simple structure, strong tissue infiltration, and low immunogenicity. Single-domain antibodies do not contain an Fc domain, and therefore do not mediate ADCC effects to induce cytotoxicity when used as targeting molecules. Furthermore, they exhibit good stability, resistance to high temperatures and extreme pH environments, and low production costs. Therefore, single-domain antibodies are excellent targeting molecules with significant potential for targeted drug development.

[0005] Therefore, the development of single-domain antibodies targeting 4-1BB is of great significance to this field. Summary of the Invention

[0006] This invention provides a single-domain antibody against 4-1BB and its application.

[0007] In a first aspect of the invention, an anti-4-1BB single-domain antibody is provided, the single-domain antibody having three complementarity-determining regions (CDRs) derived from the VHH chain of the amino acid sequence as shown in SEQ ID NO:1 or 13-17;

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

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

[0010] (1) CDR determined using IMGT rules:

[0011] The amino acid sequence is CDR1 as shown in SEQ ID NO:2, 18 or 19.

[0012] The amino acid sequence is CDR2 as shown in SEQ ID NO:3, and

[0013] The amino acid sequence is CDR3 as shown in SEQ ID NO:4;

[0014] (2) CDR determined using Kabat rules:

[0015] The amino acid sequence is CDR1 as shown in SEQ ID NO:5.

[0016] The amino acid sequence is CDR2 as shown in SEQ ID NO:6, and

[0017] The amino acid sequence is CDR3 as shown in SEQ ID NO:7.

[0018] In another preferred embodiment, the sequence of the VHH chain of the single-domain antibody is as shown in any one of SEQ ID NO: 1, 13, 14, 15, 16 or 17.

[0019] In another preferred embodiment, the anti-4-1BB single-domain antibody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0020] In another preferred embodiment, when the anti-4-1BB single-domain antibody is a humanized antibody, the sequence of the VHH chain of the single-domain antibody is as shown in any one of SEQ ID NO: 13, 14, 15, 16 or 17.

[0021] 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.

[0022] In another preferred embodiment, any of the amino acid sequences described above 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 4-1BB binding affinity.

[0023] 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.

[0024] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a frame region (FR) comprising the amino acid sequences of FR1, FR2, FR3, and FR4.

[0025] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0026] In another preferred embodiment, the frame region FR is of human, mouse, rabbit, or camel origin.

[0027] In another preferred embodiment, the single-domain antibody binds to human, mouse, or monkey-derived 4-1BB.

[0028] In another preferred embodiment, the antibody is a heavy chain antibody, which includes heavy chain constant regions CH2 and CH3 (Fc segment).

[0029] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0030] In another preferred embodiment, the VHH chain of the single-domain antibody targeting 4-1BB has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the amino acid sequence shown in SEQ ID NO:1.

[0031] In another preferred embodiment, the VHH chain of the anti-4-1BB single-domain antibody has one or more amino acid sequences as shown in SEQ ID NO:1, 13, 14, 15, 16 or 17.

[0032] In another preferred embodiment, the anti-4-1BB single-domain antibody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0033] In a second aspect of the invention, a humanized antibody is provided, wherein the humanized antibody is a humanized antibody of the anti-4-1BB single-domain antibody described in the first aspect of the invention.

[0034] In another preferred embodiment, the VHH chain of the humanized antibody against the 4-1BB single-domain antibody is selected from the group consisting of:

[0035] (1) The amino acid sequence of the VHH chain is shown in SEQ ID NO:13;

[0036] (2) The amino acid sequence of the VHH chain is shown in SEQ ID NO:14;

[0037] (3) The amino acid sequence of the VHH chain as shown in SEQ ID NO:15;

[0038] (4) The amino acid sequence of the VHH chain as shown in SEQ ID NO:16;

[0039] (5) The amino acid sequence is the VHH chain shown in SEQ ID NO:17.

[0040] In a third aspect of the invention, an anti-4-1BB single-domain antibody fusion protein is provided, the single-domain antibody fusion protein having a structure from the N-terminus to the C-terminus as shown in Formula Ia or Ib:

[0041] Ab1-FLP (Formula Ia);

[0042] PL-Ab1-F (Formula Ib);

[0043] In the formula,

[0044] Ab1 is the VHH chain of the anti-4-1BB single-domain antibody as described in the first aspect of the present invention;

[0045] F represents the Fc segment with no or no immunoglobulin;

[0046] L represents the connector sequence;

[0047] P represents a non-existent or therapeutic protein.

[0048] In another preferred embodiment, L is a flexible linker peptide.

[0049] In another preferred embodiment, the L sequence is as shown in (GGGS)n, where n is an integer selected from 1 to 6, preferably n is 2, 3 or 4.

[0050] In another preferred embodiment, P is a therapeutic antibody or a natural ligand.

[0051] In another preferred embodiment, the therapeutic antibody is a monoclonal antibody fragment or a monoclonal antibody fragment in tandem form, preferably scFv, tandem scFv, Fab, tandem Fab, single-domain antibody, tandem single-domain antibody or a combination thereof.

[0052] In another preferred embodiment, the therapeutic antibody targets tumor-associated antigens.

[0053] In a fourth aspect of the invention, a polynucleotide is provided that encodes the anti-4-1BB single-domain antibody described in the first aspect of the invention or the humanized antibody described in the second aspect of the invention.

[0054] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0055] 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.

[0056] In a sixth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fifth aspect of the invention, or having the polynucleotide described in the fourth aspect of the invention integrated into its genome.

[0057] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0058] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0059] In a seventh aspect of the invention, a method for generating an anti-4-1BB single-domain antibody is provided, comprising the steps of:

[0060] (a) Culturing the host cells described in the sixth aspect of the invention under conditions suitable for generating single-domain antibodies, thereby obtaining a culture or single-domain antibody fusion protein containing the said anti-4-1BB single-domain antibody; and

[0061] (b) Isolate or recover the anti-4-1BB single-domain antibody or single-domain antibody fusion protein from the culture.

[0062] In an eighth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0063] (a) A single-domain antibody against 4-1BB as described in the first aspect of the invention; and

[0064] (b) The coupling part selected from the following group: detectable marker, drug, or combination thereof.

[0065] In another preferred embodiment, the immunoconjugate is an antibody-drug conjugate.

[0066] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0067] In another preferred embodiment, the detectable marker is a chemical marker, a biological marker, or a combination thereof.

[0068] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0069] In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.

[0070] In another preferred embodiment, the drug is a small molecule drug, a biological factor, an oligonucleotide, or a combination thereof.

[0071] In another preferred embodiment, the oligonucleotide is an antisense oligonucleotide, small interfering RNA, microRNA, nucleic acid aptamer, or a combination thereof.

[0072] In another preferred embodiment, the drug is a cytotoxic drug (toxin).

[0073] 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.

[0074] In another preferred embodiment, the coupling portion is a detectable marker.

[0075] In another preferred embodiment, the detectable marker includes a radionuclide, wherein the radionuclide comprises:

[0076] (i) a detection isotope selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or

[0077] (ii) Therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0078] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, viral capsid proteins (VLPs), liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinoflavin (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0079] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0080]

[0081] in:

[0082] nAb is an anti-4-1BB single-domain antibody as described in the first aspect of the present invention;

[0083] LU represents a chemical bond or linker;

[0084] D is the coupling part;

[0085] p is the average number of conjugated portions in the immunoconjugate, and p is a value selected from 1 to 10.

[0086] In a ninth aspect of the present invention, a recombinant protein is provided, said recombinant protein having:

[0087] (i) The sequence of the VHH chain of the single-domain antibody as described in the first aspect of the present invention;

[0088] (ii) Optional Fc segment; and

[0089] (ii) Optional tag sequences to assist in expression and / or purification.

[0090] In another preferred embodiment, the tag sequence includes Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, 6His tag, or a combination thereof.

[0091] In another preferred embodiment, the recombinant protein specifically binds to the 4-1BB protein.

[0092] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0093] In a tenth aspect of the present invention, a multispecific antibody based on a mutually compatible nucleic acid backbone is provided, wherein the multispecific antibody is a polymer formed by a complex of n monomers having mutually compatible nucleic acid backbones, where n is a positive integer from 2 to 8;

[0094] The polymer includes the following monomers:

[0095] (a) A first monomer, comprising: (a1) a polypeptide element D1: an anti-4-1BB single-domain antibody according to the first aspect of the present invention that binds to the first target protein 4-1BB; and (a2) a first backbone nucleic acid single chain;

[0096] (b) a second monomer, the second monomer comprising: (b1) a polypeptide element D2: an anti-PD-L1 antibody binding to a second target protein PD-L1; and (b2) a second backbone nucleic acid single chain; and

[0097] (c) a third monomer, the third monomer comprising: (c1) a polypeptide element D3: an antibody that binds to a third target protein, wherein the third target protein includes PD-L1; and (c2) a third backbone nucleic acid single chain;

[0098] The polypeptide elements D1, D2, and D3 are covalently linked to the first, second, and third backbone nucleic acid single strands, respectively.

[0099] In another preferred embodiment, in the polymer, the backbone nucleic acid single strand of each monomer forms a complementary double strand with the backbone nucleic acid single strand of other monomers through base complementarity, thereby forming a complementary nucleic acid backbone complex structure.

[0100] In another preferred embodiment, in the polymer, the backbone nucleic acid single strand of each monomer forms a mutually complementary double strand with the backbone nucleic acid single strands of the other two monomers through base complementarity.

[0101] In another preferred embodiment, the polymer further includes the following monomers:

[0102] (d) A fourth monomer, the fourth monomer comprising: (d1) a half-life extension module; and (d2) a fourth backbone nucleic acid single strand.

[0103] In another preferred embodiment, the half-life extension module is covalently linked to a fourth backbone nucleic acid single strand.

[0104] In another preferred embodiment, the half-life extension module is a protein or peptide with a long half-life.

[0105] In another preferred embodiment, the half-life extension module is selected from the group consisting of: natural albumin, recombinant albumin, anti-albumin antibody, albumin-specific aptamer, protein and aptamer that directly bind FcRn, or a combination thereof.

[0106] In another preferred embodiment, the anti-albumin antibody is selected from the group consisting of nanobodies, single-chain antibodies, Fab, monoclonal antibodies, or combinations thereof.

[0107] In another preferred embodiment, the polypeptide elements D2 and D3 are each independently selected from: single-domain antibody (VHH), tandem single-domain antibody, Fab, Fab', F(ab')2, TriFab, Fv fragment, fragment variable (Fv) heterodimer, single-chain Fv (scFv) fragment, diabody, bispecific T cell conjugate (BiTE) or single-domain fragment, preferably single-domain antibody (VHH).

[0108] In another preferred embodiment, the polypeptide element D2 is an anti-PD-L1 single-domain antibody or scFv.

[0109] In another preferred embodiment, the polypeptide element D3 is an anti-PD-L1 single-domain antibody or scFv.

[0110] In an eleventh aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0111] (i) a single-domain antibody against 4-1BB as described in the first aspect of the present invention, or a fusion protein as described in the third aspect of the present invention, or an immunoconjugate as described in the eighth aspect of the present invention, or a recombinant protein as described in the ninth aspect of the present invention, or a multispecific antibody as described in the tenth aspect of the present invention, or a combination thereof; and

[0112] (ii) Pharmaceutically acceptable carriers.

[0113] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0114] In a twelfth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: a single-domain antibody against 4-1BB as described in the first aspect of the invention, a fusion protein as described in the third aspect of the invention, an immunoconjugate as described in the eighth aspect of the invention, a recombinant protein as described in the ninth aspect of the invention, a multispecific antibody as described in the tenth aspect of the invention, a pharmaceutical composition as described in the eleventh aspect of the invention, or a combination thereof, wherein the active ingredient is used for:

[0115] (a) Preparation of detection reagents, detection plates or kits; and / or

[0116] (b) To prepare medicines for the prevention and / or treatment of diseases.

[0117] In another preferred embodiment, the disease is a tumor.

[0118] In another preferred embodiment, the tumor is a solid tumor or a non-solid tumor.

[0119] In another preferred embodiment, the tumor is selected from the group consisting of: chronic lymphocytic leukemia, non-Hodgkin's lymphoma, hematologic malignancies, bladder cancer, urothelial carcinoma, recurrent head and neck squamous cell carcinoma, advanced neurosecretory carcinoma, non-small cell lung cancer, esophageal adenocarcinoma, melanoma, or combinations thereof.

[0120] In another preferred embodiment, the detection reagent, detection plate, or kit is used for:

[0121] (1) Detect 4-1BB protein in the sample; and / or

[0122] (2) Detection of cells expressing 4-1BB protein.

[0123] In another preferred embodiment, the test reagent, test plate, or kit is used to diagnose 4-1BB-related diseases.

[0124] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0125] In another preferred embodiment, the active ingredient is also used for one or more of the following purposes:

[0126] (i) Used for the detection of human 4-1BB molecules;

[0127] (ii) Used for flow cytometry detection;

[0128] (iii) Used for cell immunofluorescence detection;

[0129] (iv) Used to treat tumors.

[0130] In a thirteenth aspect of the present invention, a method for detecting 4-1BB protein in a sample is provided, the method comprising the steps of:

[0131] (1) Contact the sample with a single-domain antibody against 4-1BB as described in the first aspect of the present invention;

[0132] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of 4-1BB protein in the sample.

[0133] In a fourteenth aspect of the present invention, a 4-1BB protein detection reagent is provided, the detection reagent comprising:

[0134] (i) the single-domain antibody against 4-1BB as described in the first aspect of the present invention, or the immunoconjugate as described in the eighth aspect of the present invention, or the recombinant protein as described in the ninth aspect of the present invention; and

[0135] (ii) A detectable carrier.

[0136] In another preferred embodiment, the coupling portion of the immunoconjugate is a detection isotope.

[0137] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0138] 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.

[0139] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0140] In another preferred embodiment, the test reagent is in liquid or powder form (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, or inhaler).

[0141] In a fifteenth aspect of the present invention, a detection kit for 4-1BB protein is provided, the kit containing the immunoconjugate described in an eighth aspect of the present invention or the detection reagent described in a fourteenth aspect of the present invention, and an instruction manual.

[0142] In another preferred embodiment, the specification states that the kit is used for non-invasive detection of 4-1BB expression in a test subject.

[0143] In a sixteenth aspect of the invention, a method for treating a disease is provided, the method comprising administering to a desired subject a single-domain antibody against 4-1BB as described in the first aspect of the invention, or an immunoconjugate as described in the eighth aspect of the invention, or a recombinant protein as described in the ninth aspect of the invention, or a multispecific antibody as described in the tenth aspect of the invention, or a pharmaceutical composition as described in the eleventh aspect of the invention.

[0144] In another preferred embodiment, the object includes a human or a non-human mammal.

[0145] In another preferred embodiment, the non-human mammals include rodents (such as mice and rabbits) and non-human primates (such as monkeys).

[0146] In another preferred embodiment, the disease is a tumor.

[0147] In another preferred embodiment, the tumor is a solid tumor or a non-solid tumor.

[0148] In another preferred embodiment, the tumor is selected from the group consisting of: chronic lymphocytic leukemia, non-Hodgkin's lymphoma, hematologic malignancies, bladder cancer, urothelial carcinoma, recurrent head and neck squamous cell carcinoma, advanced neurosecretory carcinoma, non-small cell lung cancer, esophageal adenocarcinoma, melanoma, or combinations thereof.

[0149] 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

[0150] Figure 1 The flow cytometry analysis showed positive monoclonal antibodies against 4-1BB binding to the antigen in yeast.

[0151] Figure 2 The flow cytometry results demonstrate the binding ability of the anti-4-1BB single-domain antibody to h4-1BB CHO-K1 cells.

[0152] Figure 3 This demonstrates the ability of ELISA to detect the binding of F9 to human 4-1BB antigen.

[0153] Figure 4 This demonstrates the ability of ELISA to detect the binding of F9 to monkey 4-1BB antigen.

[0154] Figure 5 The specificity of F9 binding was demonstrated by flow cytometry.

[0155] Figure 6 The analysis of F9's ligand blocking activity is shown.

[0156] Figure 7 A schematic diagram of the F9 / PD-L1 bispecific antibody and an SDS-PAGE electrophoresis gel image are shown.

[0157] Figure 8 The activation activity of the F9 / PD-L1 bispecific antibody on h4-1BB / NF-κB-Luc2P 293T reporter cells is shown.

[0158] Figure 9 Analysis of the binding activity of the humanized F9 sequence with h4-1BB CHO-K1 cells was shown. Detailed Implementation

[0159] Through extensive and in-depth research and screening, the inventors have developed for the first time a single-domain antibody targeting 4-1BB and its humanized antibody. This single-domain antibody does not bind to OX40 and GITR, which belong to the TNFRSF family, and does not affect the binding of 4-1BB to 4-1BBL. Furthermore, based on this antibody, the inventors designed and constructed multispecific antibodies (such as a 4-1BB / PD-L1 bispecific antibody). Experimental results show that the single-domain antibody and its humanized antibody obtained in this invention have specific high affinity and binding activity to 4-1BB, and good stability. The single-domain antibody of this invention exhibits cross-binding activity between human and monkey species. Moreover, the 4-1BB / PD-L1 bispecific antibody of this invention can effectively activate the 4-1BB signaling pathway. Based on these findings, this invention was completed.

[0160] the term

[0161] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0162] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0163] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0164] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0165] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0166] 4-1BB

[0167] 4-1BB, also known as CD137 / TNFRSF9, is a member of the tumor necrosis factor receptor superfamily. It is a type I transmembrane protein composed of an extracellular, transmembrane, and intracellular region. It is primarily expressed on the surface of activated T cells and is a T cell co-stimulatory molecule; its binding to the ligand 4-1BBL stimulates T cell activation and proliferation. Anti-4-1BB monoclonal antibodies selectively regulate CD8+. + The proliferation of T cells and the expression of the pro-inflammatory cytokine IFNγ promote tumor clearance and have anti-tumor activity.

[0168] Anti-4-1BB single-domain antibody

[0169] As used herein, the terms "anti-4-1BB single-domain antibody of the present invention," "single-domain antibody of the present invention," "4-1BB single-domain antibody of the present invention," "anti-4-1BB nanobody of the present invention," "nanobody of the present invention," and "4-1BB nanobody of the present invention" are used interchangeably and all refer to single-domain antibodies that specifically recognize and bind to 4-1BB. Particularly preferred are single-domain antibodies with the amino acid sequence of the VHH chain as shown in SEQ ID NO:1 or any of 13-17.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] As those skilled in the art will recognize, 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-4-1BB protein single-domain antibody or fragments thereof.

[0174] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0175] As used in this article, the terms “complementarity determining region” and “CDR” are used interchangeably.

[0176] 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.

[0177] 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.

[0178] 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 4-1BB proteins, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0179] 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 (e.g., immunoconjugate and fusion protein) having a heavy chain containing a variable region, provided that the variable region is the same as or at least 90% homologous to the heavy chain variable region of the antibody of the present invention, preferably at least 95% homologous.

[0180] This invention includes not only complete antibodies, but also fragments, derivatives and analogs of said antibodies.

[0181] 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.

[0182] The antibody of this invention refers to a polypeptide having 4-1BB 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.

[0183] 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.

[0184] The present invention also provides other polypeptides, such as fusion proteins comprising a single-domain antibody or a fragment thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the single-domain antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 80 consecutive amino acids, and more preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0185] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0186] Table A

[0187]

[0188]

[0189] The single-domain antibody of this invention specifically binds to 4-1BB without binding to OX40 and GITR, which belong to the same TNFRSF family, and does not affect the binding of 4-1BB to 4-1BBL. In one embodiment, the single-domain antibody of this invention exhibits cross-reactivity in human and monkey-derived 4-1BB.

[0190] Polynucleotides, vectors and host cells

[0191] 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.

[0192] 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.

[0193] 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.

[0194] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, in which the additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Typically, expression vectors useful in recombinant DNA technologies are in the form of plasmids.

[0195] As used herein, the term "antigen" or "target antigen" refers to a molecule or part of a molecule that can be bound by an antibody or antibody-like binding protein. The term further refers to a molecule or part of a molecule that can be used in animals to produce an antibody capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. For each target antigen recognized by an antibody or by an antibody-like binding protein, the antibody-like binding protein can compete with the intact antibody that recognizes the target antigen.

[0196] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for both the light and heavy chain domains to fold into an exchangeable dual variable region immunoglobulin. Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element to be achieved in the linker. Preferred linkers may be (GS)n, (G3S)n, or (G4S)n (n selected from 1-6).

[0197] As used herein, a “variant” of an antibody, antibody fragment, or antibody domain means that the antibody, antibody fragment, or antibody domain: (1) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target that specifically binds to the original antibody, antibody fragment, or antibody domain. It should be understood that when sequence identity is expressed in the form of “at least x% identical” or “at least x% identity,” such embodiments include any and all numerical percentages equal to or higher than the lower limit. Furthermore, it should be understood that the presence of an amino acid sequence in this application should be interpreted as additional disclosure or inclusion of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with that amino acid sequence.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] Immunoconjugates

[0211] The present invention also provides immunoconjugates based on the antibodies of the present invention, preferably nanobody-drug conjugates (NDCs).

[0212] 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 drug, a small molecule drug, an agonist small molecule (STING, TLR7, TLR8, etc.), an oligonucleotide, or a radionuclide.

[0213] 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).

[0214] 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.

[0215] Single-domain antibodies can be conjugated to drugs to form single-domain 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.

[0216] 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 polyethylene glycol-modified SMCC crosslinking agent (SM(PEG)2), or a maleimide attached to the antibody via a thiosuccinimide.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] The immunoconjugated drug of the present invention can also be an antibody-oligonucleotide conjugate (AOC), which is composed of a single-domain antibody of the present invention conjugated with a therapeutic oligonucleotide. Oligonucleotides that can be used in the AOC of the present invention include, but are not limited to, antisense oligonucleotides, small interfering RNA, microRNAs, and nucleic acid aptamers.

[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 the single-domain antibody conjugate NDC, is shown in the following molecular formula:

[0226]

[0227] in:

[0228] nAb refers to the aforementioned single-domain antibodies targeting SA, heavy-chain antibodies targeting SA, or multispecific antibodies.

[0229] LU stands for connector / connector;

[0230] D is the coupling part;

[0231] And the subscript p is a value selected from 1 to 10.

[0232] Pharmaceutical Composition

[0233] 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.

[0234] The pharmaceutical compositions of the present invention may contain any antitumor drug (such as an antitumor antibody) linked to the anti-4-1BB single-domain antibody of the present invention, and thus may be used to treat tumors. Furthermore, other therapeutic agents may be used simultaneously.

[0235] 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 single-domain antibody (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.

[0236] 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.

[0237] Labeled single-domain antibodies

[0238] In a preferred embodiment of the invention, the single-domain antibody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0239] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the invention, a single-domain antibody against 4-1BB is labeled with colloidal gold to obtain a colloidal gold-labeled single-domain antibody.

[0240] Multispecific antibodies

[0241] As used herein, the term "multispecific antibody" refers to a molecule comprising at least two targeting domains with different binding specificities, wherein at least one targeting domain specifically binds to NK cell surface antigens. In some embodiments, multispecific inhibitors are polypeptides comprising a scaffold and two or more immunoglobulin antigen-binding domains targeting different antigens or epitopes. In some embodiments, multispecific antibodies are bispecific or trispecific antibodies.

[0242] As used herein, the terms "bispecific antibody of the present invention," "bispecific fusion protein of the present invention," and "2Sbody" are used interchangeably and refer to molecules comprising two targeting domains having two different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, upon binding to the target molecule, inhibiting the biological function of the target molecule. In some embodiments, the bispecific antagonist is a polymer molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen-binding domain or CDR of the antibody. In some embodiments, the targeting domain comprises a ligand or fragment thereof that specifically binds to a target protein.

[0243] It should be understood that the bispecific fusion protein of the present invention is essentially a bispecific antibody, an antibody molecule capable of simultaneously and specifically binding to two antigens (4-1BB and PD-L1). Based on symmetry, bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules.

[0244] The single-chain nucleic acid in the multispecific antibody of this invention is a nucleic acid chain that is resistant to degradation in vivo and does not elicit a strong innate immune response. Typically, the single-chain nucleic acid includes, but is not limited to, L-type nucleic acid, peptide nucleic acid, locked nucleic acid, morpholine phosphate nucleic acid, L-morpholine phosphate nucleic acid, thio-modified nucleic acid, 2'-fluoro-modified nucleic acid, and 5-hydroxymethylcytosine nucleic acid.

[0245] The targeting molecule can be any targeting antibody, ligand, or small molecule that specifically binds to the target. The targeting antibody can be a monoclonal antibody or its antigen-binding fragment. In one embodiment, the targeting antibody can be a single-domain antibody targeting 4-1BB, for example, having the sequence shown in SEQ ID NO:1 or any of 13-17. The ligand can be a natural ligand or its targeting variant.

[0246] In a preferred embodiment of the present invention, the multispecific antibody comprises four morpholine phosphate oligonucleotides with different sequences, which can precisely self-assemble into a stable tetrameric nucleic acid backbone.

[0247] Detection methods

[0248] The present invention also relates to a method for detecting 4-1BB protein. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of 4-1BB protein in the dissolved samples.

[0249] 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.

[0250] Reagent test kit

[0251] 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.

[0252] This invention also provides a detection kit for detecting 4-1BB levels, comprising an antibody that recognizes the 4-1BB 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.

[0253] application

[0254] As described above, the single-domain antibody of the present invention or the multispecific fusion antibody containing the single-domain antibody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of diseases related to 4-1BB, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of 4-1BB.

[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) The production of the single-domain antibody of the present invention is simple.

[0258] (c) The single-domain antibody of the present invention specifically binds to 4-1BB but does not bind to 4-1BB family proteins OX40 and GITR.

[0259] (d) This invention provides a single-domain antibody with cross-binding activity between human and monkey species, and the antibody does not block the binding of 4-1BB ligand.

[0260] (e) The present invention is based on the design of a 4-1BB / PD-L1 multispecific antibody against a single domain antibody, which can effectively activate NK cells and effectively kill tumor cells; it has great potential for the development of anti-tumor drugs.

[0261] 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.

[0262] Example 1: Screening of anti-4-1BB single-domain antibodies

[0263] A yeast display library of artificially synthesized single-domain antibodies was amplified for screening 4-1BB single-domain antibodies. Eighty-eight positive yeast clones were selected through three rounds of protein-based magnetic bead sorting, four rounds of protein-based flow cytometry sorting, and three rounds of cell-based flow cytometry sorting. Sequencing analysis was performed on the clones, and after protein translation, amino acid sequence alignment and phylogenetic tree construction were conducted. The binding activity of the clones was verified based on sequence alignment and flow cytometry analysis. Figure 1 A total of 8 unique human 4-1BB antigen-binding sequences were obtained.

[0264] Example 2 Construction of yeast strain expressing anti-4-1BB single-domain antibody and protein preparation

[0265] A his-flag tag was added to the N-terminus of the anti-4-1BB single-domain antibody, and a "GSC" tripeptide was added to the C-terminus. 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. 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 be secreted and expressed 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.

[0266] The protein expression and purification methods are as follows: 400 μL of bacterial culture was inoculated into 200 mL of BMGY medium and cultured at 30°C 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 for 3 days at 20°C and 250 rpm. After induction, the supernatant was collected by centrifugation at 12000 rpm for 15 min using 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 histagged 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.

[0267] The expression levels of the 8 unique sequences in a 100ml culture system are summarized in Table 1. Except for A5, the other 7 antibodies can be expressed normally in the yeast system, with F9 showing the highest expression level.

[0268] Table 1 Summary of yeast expression levels of anti-4-1BB single-domain antibodies

[0269]

[0270]

[0271] The antibody sequence of F9 is shown in SEQ ID NO:1, and its CDR sequences determined by the IMGT rule and the Kabat rule are shown in Table 2.

[0272] QVQLQESGGGLVQAGGSLRLSCAAS GNISYQPI MGWYRQAPGKEREFV AS IDSGGIT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AAWTY VENEYIVDHWY WGQGTQVTVSS(SEQ ID NO:1).

[0273] Table 2

[0274] CDR determined by IMGT rules SEQ ID NO: Sequence information CDR1 2 GNISYQPI CDR2 3 IDSGGIT CDR3 4 AAWTYVENEYIVDHWY CDR determined by Kabat rules SEQ ID NO: Sequence information CDR1 5 QPIMG CDR2 6 SIDSGGITYYADSVKG CDR3 7 WTYVENEYIVDHWY

[0275] Example 3: Binding activity analysis of anti-4-1BB single-domain antibody with human 4-1BB-overexpressing CHO-K1

[0276] 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 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 minutes. Centrifuge at 1000 rpm for 5 minutes at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, add 200 μL / well of 1:1000 diluted FITC-labeled mouse anti-flag tag antibody (Genscript, Cat#A01632) solution, vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm for 5 minutes 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.

[0277] The mean fluorescence intensity of each sample was determined using flow cytometry (BD, FACSCelesta). The binding EC50 value was calculated by fitting an S-curve with a 4-parameter equation using GraphPadPrism 10 software.

[0278] Experimental results showed that, except for F9, most candidate antibodies had weak binding affinity to 4-1BB on the cell surface. Figure 2 ).

[0279] Example 4: Binding activity analysis of F9 with human and monkey 4-1BB recombinant protein

[0280] Prepare human 4-1BB-His (ACRO, Cat#41B-H52HC) antigen solution and monkey 4-1BB-His (ACRO, Cat#41B-C52H4) antigen solution with PBS to a final concentration of 1 μg / mL. Add 100 μL / well to each well of a 96-well ELISA plate and incubate overnight at 4°C. Wash three times with PBST (PBS + 0.05% Tween 20), add 200 μL / well of blocking buffer (PBST + 3% B4-1BB), and block at room temperature for 1 hour. Wash three times with PBST, add 100 μL / well of serially diluted single-domain antibody solution, and incubate at room temperature for 1 hour. Wash three times with PBST, add 100 μL / well of 1:5000 diluted horseradish peroxidase-labeled rabbit anti-camel VHH antibody (Genscript, Cat#A02016), and incubate at room temperature for 1 hour. Wash three times with PBST, blot dry, add 100 μL / well of TMB substrate solution (Beyotime, Cat#P0209), and incubate at room temperature for 5 to 30 minutes. Then add 100 μL / well of stop solution (Beyotime, Cat#P0215).

[0281] The absorbance at 450 nm in each well was measured using a Molecular Devices (SpectraMax i3x) microplate reader. The binding EC50 values ​​were calculated by fitting an S-curve using a 4-parameter equation in GraphPad Prism 10 software.

[0282] Experimental results show that candidate antibody F9 has cross-binding activity between humans and monkeys. Figure 3-4 ).

[0283] Example 5F9 does not bind to OX40 and GITR, which belong to the same TNFRSF family.

[0284] h4-1BB CHO-K1, hOX40 CHO-K1, and hGITR CHO-K1 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. 6Cells / mL, add 180 μL / well of cell suspension to a 96-well U-shaped plate. Prepare a 1 μM antibody solution using flow cytometry buffer. 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 minutes. Centrifuge at 1000 rpm for 5 minutes at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, add 200 μL / well of 1:1000 diluted FITC-labeled mouse anti-flag tag antibody (Genscript, Cat#A01632) solution, vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm for 5 minutes 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.

[0285] The average fluorescence intensity of each sample was determined using a flow cytometer (BD, FACSCelesta).

[0286] Experimental results showed that, except for F9 and the control antibody which specifically bind to h4-1BB CHO-K1, but not to hOX40 CHO-K1 or hGITR CHO-K1, the binding of F9 to 4-1BB was specific. Figure 5 ).

[0287] Example 6F9 does not affect the bonding between 4-1BB and 4-1BBL.

[0288] Prepare a human 4-1BB-His (ACRO, Cat#41B-H52HC) antigen solution with PBS to a final concentration of 1 μg / mL. Add 100 μL / well to each well of a 96-well ELISA plate and incubate overnight at 4°C. Wash three times with PBST (PBS + 0.05% Tween 20), add 200 μL / well of blocking buffer (PBST + 3% BSA), and block at room temperature for 1 hour. Wash once with PBST, add 50 μL / well of serially diluted F9 antibody solution, and incubate at room temperature for 30 minutes. Add 50 μL / well of human 4-1BBL-Fc (ACRO, Cat#41L-H5269) solution to a final concentration of 5 nM and incubate at room temperature for 1 hour. Wash three times with PBST, add 100 μL / well of a 1:5000 diluted horseradish peroxidase-labeled goat anti-human IgG antibody (Yeasen, Cat#33501ES60), and incubate at room temperature for 1 hour. Wash three times with PBST, blot dry, add 100 μL / well of TMB substrate solution (Beyotime, Cat#P0209), and incubate at room temperature for 5 to 30 minutes. Then add 100 μL / well of stop solution (Beyotime, Cat#P0215).

[0289] The absorbance at 450 nm in each well was measured using a Molecular Devices (SpectraMax i3x) microplate reader. The binding EC50 values ​​were calculated by fitting an S-curve using a 4-parameter equation in GraphPad Prism 9 software.

[0290] Experimental results show that candidate antibody F9 does not block the binding of h4-1BB to ligand h4-1BBL. Figure 6 ).

[0291] Example 7: Preparation of F9 / PD-L1 Bispecific Antibody

[0292] The purified C-terminal "GSC" tripeptide F9 from Example 2 was reduced, exposing the thiol group of the C-terminal cysteine ​​of the reduced F9 single-domain antibody. Subsequently, it was coupled with maleimide-modified L-DNA1 and L-DNA2 and purified to obtain F9-L-DNA1 and F9-L-DNA2.

[0293] Anti-PD-L1 single-domain antibody nucleic acid conjugates PD-L1-L-DNA3 and PD-L1-L-DNA4 were prepared using the same method.

[0294] The nucleic acid assembly modules of the conjugated nanobody were mixed in a molar ratio of 1:1:1:1 to obtain the 4-1BB / PD-L1 bispecific antibody NAPPA-F9(2)-PD-L1(2). A schematic diagram of the bispecific antibody and an SDS-PAGE electrophoresis image are shown below. Figure 7 .

[0295] The sequence of the anti-PD-L1 single-domain antibody is as follows (from patent US2017 / 0198050 A1, hz28A2V5):

[0296] ENLYFQSEVQLLESGGGEVQPGGSLRLSCAASGGIFAIKPISWYRQAPGK QREWVSTTTSSGATNYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYY CNVFEYWGQGTLVTVKP(SEQ ID NO:8)

[0297] The L-DNA nucleic acid sequence is as follows:

[0298] L-DNA1: 5'AGGCGATCACAATCCAAATGAGCGTGTTACGG 3' (SEQ ID NO: 9);

[0299] L-DNA2: 5'ACCGTAACACGCTCAAAACCGAAGTGCCAATT 3' (SEQ ID NO: 10);

[0300] L-DNA3: 5'AAATTGGCACTTCGGAAAACTATGCGGCTGCT 3' (SEQ ID NO: 11);

[0301] L-DNA4: 5'AAGCAGCCGCATAGTAAAGGATTGTGATCGCC 3' (SEQ ID NO: 12).

[0302] Example 8: F9 / PD-L1 possesses target cell-dependent 4-1BB signaling pathway activation activity.

[0303] 293T cells expressing h4-1BB and NF-κB-driven luciferase reporter genes (h4-1BB / NF-κB-Luc2P293T) were used as effector cells and seeded into cell culture plates at a density of 40,000 cells / 50 μL / well, with triplet wells, and incubated overnight at 37°C. Serially diluted 10-fold concentrations of NAPPA-F9(1,2)-PD-L1(3,4) solutions were prepared, and 10 μL / well of the solution was added to each cell culture plate for pre-incubation at 37°C for 15 minutes. hPD-L1-positive CHO-K1 cells (hPD-L1 CHO-K1) and hPD-L1-negative host cells (CHO-K1) were used as target cells and seeded into cell culture plates at a density of 40,000 cells / 40 μL / well. These cells were then mixed with effector cells pre-incubated with NAPPA-F9(1,2)-PD-L1(3,4). The cell culture plates were incubated at 37°C for 5 hours. Cells were treated with a luciferase reporter gene assay kit (Novizan, DD1203), and the chemiluminescence intensity of each well was measured using a microplate reader.

[0304] The EC50 value was calculated by fitting an S-curve with a 4-parameter equation using GraphPad Prism 10 software. Figure 8 ).

[0305] The experimental results showed that 4-1BB activation was weaker in hPD-L1-negative cells and stronger in hPD-L1-positive cells, indicating that NAPPA-F9(1,2)-PD-L1(3,4) activation of the 4-1BB signaling pathway is PD-L1-dependent.

[0306] Example 9: Humanization of Anti-4-1BB Single-Domain Antibody

[0307] Humanization of the anti-4-1BB single-domain antibody was achieved by comparing the parent sequence with the human Germline database, defining the CDR and framework regions of the parent antibody, and designing sequences with different degrees of humanization based on the differences in the framework regions (Table 3). The CDR1 of some sequences was optimized by removing glycosylation sites (Table 4).

[0308] Table 3 Summary of Humanized Sequences of Anti-4-1BB Single-Domain Antibodies

[0309]

[0310] Note: The underlined portions of each sequence represent CDR1, CDR2, and CDR3 as determined by the IMGT rules. The underlined and bolded portions in the sequences represent CDR1 optimized by removing glycosylation sites.

[0311] Table 4. CDR1 sequences after glycosylation site removal.

[0312] CDR determined by IMGT rules SEQ ID NO: Sequence information CDR1 18 GNIQYQPI CDR1 19 GNINYQPI

[0313] Example 9: Binding activity analysis of humanized F9 to human 4-1BB protein

[0314] The humanized and unhumanized F9 single-domain antibodies were expressed using a yeast expression system and purified by nickel ion affinity chromatography. The expression and purification methods were the same as in Example 2.

[0315] The binding activity of humanized F9 to human 4-1BB protein (ACRO, Cat#41B-H52HC) was analyzed by surface plasmon resonance (SPR), with unhumanized F9 serving as a control. The instrument used was a biacore T200 (Cytiva), and the binding curves were fitted using a steady-state fitting method.

[0316] The test results are shown in Table 5. All humanized VHHs bound human 4-1BB-His, and the binding activity was comparable to that of the parent strain. VHH1, VHH2, and VHH3 were slightly stronger than the parent strain.

[0317] Table 5. SPR test results of anti-4-1BB humanized VHH-Fc

[0318]

[0319] Example 10: Binding activity analysis of humanized F9 cells with high expression of human 4-1BB

[0320] h4-1BB CHO-K1 cells in logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS), and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 5Cells / 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).

[0321] The S-curve was fitted with a 4-parameter equation using GraphPad Prism 10 software, and the combined EC50 value was calculated. Figure 9 ).

[0322] Experimental results showed that all humanized F9 cells bound to h4-1BB CHO-K1 cells, and their binding activity was superior to that of the parent F9 cells.

[0323] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A single-domain antibody against 4-1BB, characterized in that, The single-domain antibody has three complementarity-determining regions (CDRs) derived from the VHH chain shown in SEQ ID NO:1 or 13-17. The CDRs are CDR1, CDR2, and CDR3 determined by any one of the IMGT rule, Kabat rule, Chothia rule, or AbM rule.

2. The single-domain antibody as described in claim 1, characterized in that, CDR1, CDR2 and CDR3 are selected from the following group: (1) CDR determined using IMGT rules: The amino acid sequence is CDR1 as shown in SEQ ID NO:2, 18 or 19. The amino acid sequence is CDR2 as shown in SEQ ID NO:3, and The amino acid sequence is CDR3 as shown in SEQ ID NO:4; (2) CDR determined using Kabat rules: The amino acid sequence is CDR1 as shown in SEQ ID NO:

5. The amino acid sequence is CDR2 as shown in SEQ ID NO:6, and The amino acid sequence is CDR3 as shown in SEQ ID NO:

7.

3. A polynucleotide, characterized in that, The polynucleotide encodes the anti-4-1BB single-domain antibody of claim 1.

4. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 3.

5. A host cell containing the expression vector of claim 4, or having the polynucleotide of claim 3 integrated into its genome.

6. A method for generating a single-domain antibody against 4-1BB, comprising the steps of: (a) Culturing the host cells of claim 5 under conditions suitable for generating single-domain antibodies to obtain a culture or single-domain antibody fusion protein containing the said anti-4-1BB single-domain antibody; and (b) Isolate or recover the anti-4-1BB single-domain antibody or single-domain antibody fusion protein from the culture.

7. An immunoconjugate comprising: (a) the single-domain antibody against 4-1BB as described in claim 1; and (b) The coupling part selected from the following group: detectable marker, drug, or combination thereof.

8. A multispecific antibody based on a mutually compatible nucleic acid backbone, characterized in that, The multispecific antibody is a polymer formed by n monomers with mutually compatible nucleic acid backbones, where n is a positive integer from 2 to 8; The polymer includes the following monomers: (a) A first monomer comprising: (a1) a polypeptide element D1: an anti-4-1BB single-domain antibody of claim 1 that binds to the first target protein 4-1BB; and (a2) a first backbone nucleic acid single chain; (b) a second monomer, the second monomer comprising: (b1) a polypeptide element D2: an anti-PD-L1 antibody binding to a second target protein PD-L1; and (b2) a second backbone nucleic acid single chain; and (c) a third monomer, the third monomer comprising: (c1) a polypeptide element D3: an antibody that binds to a third target protein, wherein the third target protein includes PD-L1; and (c2) a third backbone nucleic acid single chain; The polypeptide elements D1, D2, and D3 are covalently linked to the first, second, and third backbone nucleic acid single strands, respectively.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the single-domain antibody against 4-1BB as claimed in claim 1, or the immunoconjugate as claimed in claim 7, or the multispecific antibody as claimed in claim 8, or a combination thereof; and (ii) Pharmaceutically acceptable carriers.

10. The use of an active ingredient, characterized in that, The active ingredient is selected from the group consisting of: the anti-4-1BB single-domain antibody as described in claim 1, or the immunoconjugate as described in claim 7, or the multispecific antibody as described in claim 8, or the pharmaceutical composition as described in claim 9, or a combination thereof, wherein the active ingredient is used for: (a) Preparation of detection reagents, detection plates or kits; and / or (b) To prepare drugs for the prevention and / or treatment of tumors.

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