A single-domain antibody targeting 4-1BB, its fusion protein, its pharmaceutical composition and use

Abstract

To provide a more efficient antibody that targets 4-1BB. [Solution] The anti-4-1BB single-domain antibody is an anti-4-1BB single-domain antibody that includes a heavy chain variable region, the heavy chain variable region including CDR1 to CDR3, CDR1 having the amino acid sequence of SEQ ID NO: 79, CDR2 having the amino acid sequence of SEQ ID NO: 119, and CDR3 having the amino acid sequence of SEQ ID NO: 159.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to single-domain antibodies targeting 4-1BB, their fusion proteins, their pharmaceutical compositions and uses.

Background Art

[0002] Tumor necrosis factor receptor superfamily member 4-1BB, also known as CD137 or TNFRF9, is a member of the TNF receptor family. 4-1BB is a 255-amino acid type I transmembrane protein (NCBI: NP_001552) composed of an N-terminal signal peptide containing 17 amino acids, an extracellular region of 169 amino acids, a transmembrane region of 27 amino acids, and a C-terminal intracellular region of 42 amino acids. 4-1BB is mainly expressed in activated T cells, NK cells, regulatory T cells, dendritic cells, monocytes, neutrophils and eosinophils, and it has also been reported that endothelial cells of tumor blood vessels express 4-1BB.

[0003] During the T cell activation process, the 4-1BB molecule can provide costimulatory signals to T cells. When the T cell receptor comes into contact with an antigen, 4-1BB expression increases, and the binding of 4-1BB to the ligand activates the NF-κB signaling pathway, resulting in the activation and proliferation of T cells. However, 4-1BB can also inhibit apoptosis of activated cells. Anti-4-1BB monoclonal antibodies have been confirmed to have antitumor activity in animal models and in vitro experiments. Anti-4-1BB monoclonal antibodies selectively induce the proliferation of CD8+ T cells, upregulate the expression of pro-inflammatory cytokines such as IFN-γ, enhance the killing effect of antigen-specific effector T cells, and thereby promote tumor clearance. Anti-4-1BB monoclonal antibodies can also induce the proliferation of NK cells, thereby increasing the cytotoxic activity of CD8+ T cells. Anti-4-1BB antibodies can also induce vascular endothelial cells in tumor cells, upregulating the expression of adhesion molecules and promoting the infiltration of activated T lymphocytes into tumor tissue. Furthermore, anti-4-1BB antagonist antibodies can also alleviate autoimmune diseases such as autoimmune encephalomyelitis, lupus-like syndrome, and collagen-induced arthritis in animal models.

[0004] Therefore, 4-1BB is an important potential target in the treatment of tumors and several autoimmune diseases. Specifically, in preclinical animal models of colorectal cancer, lung cancer, breast cancer, and melanoma, agonist molecules targeting 4-1BB have shown significant antitumor activity, either as monotherapy or in combination with anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-HER-2, and other antibodies (Etxeberria I, et al. ESMO Open 2020; 4:e000733). Currently, two 4-1BB antibodies, namely urelumab (BMS-663513, developed by Bristol-Myers Squibb) and utomirumab (PF-05082566, developed by Pfizer), are in clinical trials. While urelumab has been shown in studies to be a potent superagonist, it also has side effects such as hepatotoxicity and fatigue. On the other hand, utomirumab has an advantage in terms of safety, but its agonist activity is limited compared to urerumab. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, in this field, there is an urgent need for the development of more efficient antibodies and related drugs that target 4-1BB. [Means for solving the problem]

[0006] The inventors developed anti-human 4-1BB nanobodies through intensive research and creative work. Specifically, they immunized llamas with human-derived 4-1BB antigen protein to obtain a high-quality immunonanobody gene library. From this library, the inventors screened the immunonanobody gene library to obtain two strains of anti-4-1BB nanobodies that can simultaneously bind to human and cynomolgus monkey 4-1BB and require external crosslinking for activation. Next, the two nanobodies were subjected to sequence optimization, genetically engineered mutants were expressed and purified, and further screening was performed in terms of antibody affinity, cross-binding with cynomolgus monkey 4-1BB, and ability to activate T cells to obtain nanobody strains that can be highly expressed in vitro and have high specificity. From the experimental results, it can be seen that the anti-4-1BB nanobody and fusion protein of the present invention has a small molecular weight, can cross-bind with cynomolgus monkey 4-1BB, does not block the natural interaction between the 4-1BB ligand and 4-1BB, and has strong T cell activation and good safety. Accordingly, the following invention is provided.

[0007] One aspect of the present invention relates to an anti-4-1BB single-domain antibody comprising a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 to CDR3, CDR1 having an amino acid sequence selected from SEQ ID NOs. 41 to 80, CDR2 having an amino acid sequence selected from SEQ ID NOs. 81 to 120, and CDR3 having an amino acid sequence selected from SEQ ID NOs. 121 to 160.

[0008] One aspect of the present invention relates to an anti-4-1BB single-domain antibody containing a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 to CDR3, each having one of the amino acid sequences shown in items 1 to 40 below. [Table 1]

[0009] In this invention, the CDR of an anti-4-1BB single-domain antibody is defined by the IMGT numbering system (see Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009;38(suppl_1):D301-D307).

[0010] In this invention, unless otherwise specified, 4-1BB refers to human 4-1BB. In some embodiments of this invention, 4-1BB has the amino acid sequence of SEQ ID NO: 168.

[0011] In some embodiments of the present invention, in an anti-4-1BB single-domain antibody, any one, two, three, or all four of its four framework regions are humanized. Preferably, the second framework domain is humanized, and the first, third, or fourth framework domains are also modified as necessary. Preferably, the anti-4-1BB single-domain antibody has 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, or 80% or more identity with the human anti-4-1BB single-domain antibody. Preferably, in an anti-4-1BB single-domain antibody, framework region 1 has the amino acid sequence of SEQ ID NO: 170, framework region 2 has the amino acid sequence of SEQ ID NO: 171 or SEQ ID NO: 172, framework region 3 has the amino acid sequence of SEQ ID NO: 173, and framework region 4 has the amino acid sequence of SEQ ID NO: 174.

[0012] In some embodiments of the present invention, the anti-4-1BB single-domain antibody is a K against 4-1BB. Dis less than E-07, less than 5E-08, less than 4E-08, less than 3E-08, or less than 2E-08, preferably K D This is measured by ForteBio.

[0013] In some embodiments of the present invention, the anti-4-1BB single-domain antibody has a different binding site to the 4-1BB antigen compared to urelumab and utomirumab.

[0014] In some embodiments of the present invention, an anti-4-1BB single-domain antibody specifically binds to human 4-1BB and cross-binds to cynomolgus monkey 4-1BB.

[0015] In some embodiments of the present invention, the anti-4-1BB single-domain antibody does not block the binding of the 4-1BB ligand to 4-1BB in its natural state (e.g., mammals, particularly humans).

[0016] In some embodiments of the present invention, the anti-4-1BB single-domain antibody has one amino acid sequence from SEQ ID NOs: 1 to 40.

[0017] Another aspect of the present invention relates to a fusion protein comprising an anti-4-1BB single-domain antibody as described in any one of the present inventions and an Fc fragment of human IgG or a constant region of human IgG.

[0018] In some embodiments of the present invention, the fusion protein comprises an anti-4-1BB single-domain antibody as described in any one of the present inventions and an Fc fragment of human IgG or a constant region of human IgG.

[0019] In some embodiments of the present invention, the fusion protein comprises an anti-4-1BB single-domain antibody, a linker, and an Fc fragment of human IgG or a constant region of human IgG as described in any one of the present inventions.

[0020] In some embodiments of the present invention, in the fusion protein, according to the EU numbering system, the Fc fragment of human IgG or the constant region of the heavy chain of human IgG contains the L234A mutation and the L235A mutation, or the Fc fragment of IgG further contains the G237A mutation.

[0021] In some embodiments of the present invention, the fusion protein is an anti-4-1BB heavy chain antibody. A heavy chain antibody means an antibody that does not have a light chain and is currently the general name for VHH-Fc antibodies.

[0022] In some embodiments of the present invention, in the fusion protein, the Fc fragment of human IgG is the Fc fragment of human IgG1, and the constant region of the heavy chain of human IgG is the constant region of the heavy chain of human IgG1.

[0023] In some embodiments of the present invention, in the fusion protein, the Fc fragment of human IgG is the Fc fragment of human IgG1 containing the L234A mutation and the L235A mutation, and in some cases, the Fc fragment of human IgG1 further contains the G237A mutation, preferably, the Fc fragment of human IgG1 has the amino acid sequence of SEQ ID NO: 167.

[0024] In some embodiments of the present invention, in the fusion protein, the Fc fragment of human IgG or the constant region of human IgG is linked directly or via a linker to the C-terminus of an anti-4-1BB single domain antibody.

[0025] In some embodiments of the present invention, in the fusion protein, the Fc fragment of human IgG1 or the constant region of human IgG1 is linked directly or via a linker to the C-terminus of an anti-4-1BB single domain antibody.

[0026] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the anti-4-!BB single domain antibody according to any one of the present invention or the fusion protein according to any one of the present invention.

[0027] The present invention also relates to vectors containing the isolated nucleic acid molecule of the present invention.

[0028] The present invention also relates to host cells containing the isolated nucleic acid molecule or vector of the present invention.

[0029] Another aspect of the present invention relates to a method for preparing an anti-4-1BB single-domain antibody or a fusion protein as described in any one of the present inventions, comprising the steps of: culturing host cells of the present invention under appropriate conditions; and recovering the anti-4-1BB single-domain antibody or fusion protein from the cell culture.

[0030] Another aspect of the present invention relates to a conjugate comprising an antibody moiety and a coupling moiety, wherein the antibody moiety is an anti-4-1BB single-domain antibody as described in any one of the present inventions or a fusion protein as described in any one of the present inventions, and the coupling moiety is a detectable label, preferably a radioisotope, a fluorescent substance, a luminescent substance, a coloring substance or an enzyme.

[0031] Another aspect of the present invention is a kit comprising an anti-4-1BB single-domain antibody or a fusion protein as described in any one of the present invention, or a conjugate as described in any one of the present invention. Preferably, the kit further comprises a secondary antibody capable of specifically binding to a single-domain antibody or fusion protein, and optionally the secondary antibody further comprises a detectable label such as a radioisotope, fluorescent substance, luminescent substance, colorant, or enzyme.

[0032] Another aspect of the present invention relates to the use of an anti-4-1BB single-domain antibody or a fusion protein according to any one of the present invention in the preparation of a kit for detecting the presence or level of 4-1BB in a sample.

[0033] Another aspect of the present invention relates to a pharmaceutical composition comprising an anti-4-1BB single-domain antibody or a fusion protein as described in any one of the present invention, or a conjugate of the present invention, and optionally comprising pharmaceutically acceptable excipients.

[0034] Another aspect of the present invention is the use of an anti-4-1BB single-domain antibody or a fusion protein or conjugate described in any one of the present invention in the manufacture of a drug for the prevention and / or treatment of malignant tumors or autoimmune diseases. Preferably, the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer. Preferably, the autoimmune disease is selected from the group consisting of autoimmune encephalomyelitis, loop-like syndrome, and collagen-induced arthritis, and the use relates to this.

[0035] Another aspect of the present invention is a method for treating and / or preventing malignant tumors or autoimmune diseases, comprising the step of administering an effective amount of an anti-4-1BB single-domain antibody described in any one of the present inventions, or a fusion protein described in any one of the present inventions, or a conjugate of the present invention, to a subject in need thereof. Preferably, the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer. Preferably, the method relates to a method in which the autoimmune disease is selected from the group consisting of autoimmune encephalomyelitis, loop-like syndrome, and collagen-induced arthritis.

[0036] In some embodiments of the present invention, the step of administering an effective amount of the single-domain antibody or fusion protein described in any one of the present inventions to a subject in need thereof is performed before or after surgical treatment and / or before or after radiotherapy.

[0037] In some embodiments of the present invention, in this method, The single-domain antibody or fusion protein of the present invention has a single dose of 0.1 to 100 mg per kilogram of body weight, preferably 4.8 to 24 mg or 1 to 10 mg, or the single-domain antibody or fusion protein of the present invention has a single dose of 10 to 1000 mg per subject, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg or 200 mg. Preferably, the drug is administered once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 10 days, once a week, once every 2 weeks, or once every 3 weeks. Preferably, the drug is administered by intravenous drip infusion or intravenous injection.

[0038] An anti-4-1BB single-domain antibody according to any one of the present invention, or a fusion protein according to any one of the present invention, or a conjugate according to the present invention, used for the treatment and / or prevention of malignant tumors or autoimmune diseases, Preferably, the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer. Preferably, the autoimmune disease is selected from the group consisting of autoimmune encephalomyelitis, loop-like syndrome, and collagen-induced arthritis.

[0039] As used herein, the term “antibody” typically refers to an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair having one “light” (L) chain and one “heavy” (H) chain. The light chains of antibodies can be classified into κ light chains and λ light chains. The heavy chains can be classified into μ, δ, γ, α, or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a “J” region of approximately 12 or more amino acids, and the heavy chain further contains a “D” region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody mediates the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues or factors such as the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into highly variable regions (called complementarity-determining regions (CDRs)), which contain scattered, more conserved regions called framework regions (FRs). Each of the VH and VL regions consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus. The variable regions (VH and VL) of each heavy / light chain pair form antibody-binding sites.The assignment of amino acids to regions or domains follows the definitions in Bethesda Md, Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987;196:901-917; Chothia et al. Nature 1989;342:878-883, or the IMGT numbering system (see the definition in Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009;38(suppl_1):D301-D307).

[0040] The term "antibody" is not limited to any specific method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0041] As used herein, the terms “mAb” and “monoclonal antibody” refer to an antibody or antibody fragment derived from a group of highly homologous antibody molecules, i.e., a group of identical antibody molecules, excluding spontaneous mutations that may occur spontaneously. mAbs are highly specific to a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal antibodies typically contain at least two different antibodies, and these different antibodies typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using hybridoma technology, first reported by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975;256(5517):495), and can also be obtained using recombinant DNA technology (see, for example, U.S. Patent No. 4,816,567).

[0042] As used herein, the term "humanized antibody" means an antibody or antibody fragment obtained by substituting all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), in which case the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. Furthermore, the performance of the antibody can be further improved or optimized by substituting some amino acid residues in the framework region (FR) of the recipient antibody with corresponding amino acid residues of a non-human antibody, or with amino acid residues of another antibody. For details on humanized antibodies, see, for example, Jones et al., Nature 1986; 321:522 525; Reichmann et al., Nature, 1988; 332:323 329; Presta, Curr. Op. Struct. Biol. 1992; 2:593-596; and Clark, Immunol. Today 2000; 21: 397-402. In some cases, the antigen-binding fragment of the antibody is a bispecific antibody, V H Domain and VL Although the domain is expressed on a single polypeptide chain, the linker used is too short to allow pairing between two domains on the same chain. Therefore, the domain is forced to pair with a complementary domain on another chain to form two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90:6444 6448 and Poljak RJ et al., Structure 1994; 2:1121 1123).

[0043] The fusion proteins described herein are protein products co-expressed in two genes by DNA recombination. Methods for generating and purifying antibodies and antigen-binding fragments are well known in the art (e.g., Cold Spring Harbor's Antibody Laboratory Technique Guide, Chapters 5-8 and Chapter 15).

[0044] As used herein, the terms “isolated” or “isolated” mean acquisition from its natural state by artificial means. If an “isolated” substance or component exists naturally, either the natural environment in which it exists has been altered, the substance has been isolated from its natural environment, or both have occurred. For example, if an unisolated polynucleotide or polypeptide exists naturally in a living animal, the same polynucleotide or polypeptide isolated in high purity from this natural state is referred to as “isolated.” The terms “isolated” or “isolated” do not exclude the presence of artificial or synthetic substances, nor the presence of other impurities that do not affect the activity of the substance.

[0045] As used herein, the term “vector” means a nucleic acid delivery medium into which polynucleotides can be inserted. A vector is called an expression vector if it can express a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements contained in the vector can be expressed in the host cell. Vectors are well known to those skilled in the art, and examples include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phages and M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector can contain various elements that control gene expression, including, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector can also contain an origin of replication.

[0046] As used herein, the term “host cell” means a cell that can be used to introduce a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast cells and Aspergillus, insect cells such as Drosophila S2 cells and Sf9 cells, and animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, and human cells.

[0047] As used herein, the term “pharmaceutically acceptable excipients” means carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredients, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples include, but are not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, phosphate buffers are a pH adjuster, but are not limited to; cationic, anionic, or nonionic surfactants such as Tween-80 are a surfactant, but are not limited to; and sodium chloride is an ionic strength enhancer, but is not limited to.

[0048] As used herein, the term “effective dose” means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective dose for preventing a disease (e.g., a tumor) means an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., a tumor), and an effective dose for treating a disease means an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who has the disease. Determining such an effective dose is well within the capabilities of those skilled in the art. For example, the effective dose used for treatment depends on the severity of the disease being treated, the usual state of the patient’s own immune system, the patient’s usual conditions such as age, weight and sex, the method of drug administration, and any other treatments administered simultaneously.

[0049] Furthermore, the present invention also relates to any one of the following items 1 to 11.

[0050] 1. Having one amino acid sequence from sequence numbers 1 to 40, or An amino acid sequence which is a derivative sequence of any one of the above-mentioned amino acid sequences, wherein 1 to 8 (preferably 1 to 5, more preferably 1 to 3) amino acid residues are added, deleted, modified and / or substituted, and which can maintain the 4-1BB binding affinity of 4-1BB nanobodies. Anti-4-1BB nanobody characterized by having a VHH chain.

[0051] 2. A fusion protein characterized by having the structure shown by formula I from the N-terminus to the C-terminus. Z1-L-Z2 (Formula I) (In the formula, Z1 represents one or more (preferably 1 to 2, more preferably 1) VHH chains of the anti-4-1BB nanobody described in item 1, Z2 represents the Fc fragment of immunoglobulin. (L represents any linker array)

[0052] 3. A polynucleotide characterized by encoding an anti-4-1BB nanobody as described in item 1 or a fusion protein as described in item 2.

[0053] 4. An expression vector characterized by containing the polynucleotide described in item 3.

[0054] 5. A host cell characterized by containing the expression vector described in item 4, or by having the polynucleotide described in item 3 incorporated into its genome.

[0055] 6. A method for generating anti-4-1BB nanobodies or fusion proteins, (a) A step of culturing the host cells described in item 5 under conditions suitable for the generation of anti-4-1BB nanobodies or fusion proteins, thereby obtaining a culture containing anti-4-1BB nanobodies or fusion proteins, (b) A step of isolating or recovering anti-4-1BB nanobodies or fusion proteins from the culture, A method characterized by including the following.

[0056] 7.(a) Anti-4-1BB nanobody as described in item 1 or fusion protein as described in item 2, (b) A coupling moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme, An immunoconjugate characterized by containing

[0057] 8. Use of the anti-4-1BB nanobody described in item 1 or the fusion protein described in item 2 in the manufacture of (a) reagents for detecting 4-1BB molecules or (b) drugs for treating tumors.

[0058] 9.(i) Anti-4-1BB nanobody as described in item 1, fusion protein as described in item 2, or immunoconjugate as described in item 7, (ii) A pharmaceutically acceptable carrier, A pharmaceutical composition characterized by containing the following:

[0059] 10.(i) The sequence of the anti-4-1BB nanobody described in item 1 or the fusion protein described in item 2, (ii) If necessary, a tag sequence useful for expression and / or purification, Recombinant protein characterized by containing [the specified ingredient].

[0060] 11. A protein complex characterized by being formed by hydrogen bonding and / or hydrophobic interaction between 4-1BB protein and anti-4-1BB antibody or its antigen-binding fragment, The hydrogen bond comprises more than four (preferably more than five, more preferably more than six, most preferably seven) sites of action selected from the group consisting of Asp at position 118, Leu at position 123, Arg at position 130, Val at position 132, Cys at position 134, Gly at position 135, and Ser at position 137 within the 4-1BB protein. Furthermore, the hydrophobic interaction has an interface of action consisting of more than two (preferably more than three, more preferably more than four) sites selected from the group consisting of Leu at position 123, Val at position 124, Val at position 133, and Pro at position 136 within the 4-1BB protein. The amino acid numbering of the 4-1BB protein is based on the numbering of SEQ ID NO: 168. Protein complex.

[0061] The present invention also relates to any one of the following first to fifteen aspects.

[0062] In a first aspect of the present invention, an anti-4-1BB nanobody having a VHH chain having one of the amino acid sequences of SEQ ID NOs: 1 to 40, An anti-4-1BB nanobody is provided, further comprising a derived sequence in which any of the above-described amino acid sequences is optionally modified, deleted, altered, and / or substituted with 1 to 8 (preferably 1 to 5, more preferably 1 to 3) amino acid residues, thereby maintaining the 4-1BB binding affinity of the 4-1BB nanobody.

[0063] In another preferred embodiment, the anti-4-1BB nanobody has a VHH chain having one of the amino acid sequences of SEQ ID NOs: 3, 35, 37, 38, 39, or 40.

[0064] In a second aspect of the present invention, a fusion protein having the structure shown by formula I from the N-terminus to the C-terminus is provided. Z1-L-Z2 (Formula I) (In the formula, Z1 represents one or more (preferably 1 to 2, more preferably 1) VHH chains of the anti-4-1BB nanobody described in the first aspect of the present invention, Z2 represents the Fc fragment of immunoglobulin. (L represents any linker array)

[0065] In another preferred embodiment, the fusion protein is a dimer, which is formed by a disulfide bond between the Fc fragments of Z2.

[0066] In other preferred embodiments, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4, preferably IgG1, IgG2, or IgG4.

[0067] In another preferred embodiment, the immunoglobulin is IgG1 or a variant thereof.

[0068] In other preferred embodiments, L has an amino acid sequence selected from the group consisting of GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or combinations thereof.

[0069] In another preferred embodiment, Z2 has the amino acid sequence of SEQ ID NO: 167.

[0070] In another preferred embodiment, Z2 has the same or substantially the same amino acid sequence as the amino acid sequence of SEQ ID NO: 167.

[0071] In other preferred embodiments, the term “substantially identical” means that up to 50 amino acids differ (preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to 3), and that the differences include amino acid substitutions, deletions, or additions.

[0072] In other preferred embodiments, the term “substantially identical” means that the amino acid sequences have at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to the corresponding amino acid sequence.

[0073] In another preferred embodiment, in formula I of the fusion protein, Z1 has one of the amino acid sequences of SEQ ID NOs: 1 to 40, L is absent, and Z2 has the amino acid sequence of SEQ ID NO: 167. Any of the above-mentioned amino acid sequences may have 1 to 8 (preferably 1 to 5, more preferably 1 to 3) amino acid residues added, deleted, modified, and / or substituted as needed, further comprising a derived sequence that can maintain 4-1BB binding affinity.

[0074] In another preferred embodiment, in formula I of the fusion protein, Z1 has one of the amino acid sequences of SEQ ID NOs: 3, 35, 37, 38, 39, or 40, L is absent, and Z2 has the amino acid sequence of SEQ ID NO: 167. Any of the above-mentioned amino acid sequences may have 1 to 8 (preferably 1 to 5, more preferably 1 to 3) amino acid residues added, deleted, modified, and / or substituted as needed, further comprising a derived sequence that can maintain 4-1BB binding affinity.

[0075] In another preferred embodiment, the fusion protein has an amino acid sequence from the N-terminus to the C-terminus that includes one of the sequences from SEQ ID NOs: 1 to 40 and the sequence of SEQ ID NO: 167.

[0076] In another preferred embodiment, the fusion protein has an amino acid sequence from the N-terminus to the C-terminus that includes one of the sequences of SEQ ID NOs: 3, 35, 37, 38, 39, or 40 and the sequence of SEQ ID NO: 167.

[0077] A third aspect of the present invention provides a polynucleotide encoding an anti-4-1BB nanobody as described in the first aspect of the present invention or a fusion protein as described in the second aspect of the present invention.

[0078] In other preferred embodiments, the polynucleotide includes DNA or RNA.

[0079] A fourth aspect of the present invention provides an expression vector comprising the polynucleotide described in the third aspect of the present invention.

[0080] A fifth aspect of the present invention provides a host cell in which an expression vector described in the fourth aspect of the present invention is included, or a polynucleotide described in the third aspect of the present invention is incorporated into the genome.

[0081] In other preferred embodiments, the host cells include prokaryotic or eukaryotic cells.

[0082] In other preferred embodiments, the host cells are selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0083] A sixth aspect of the present invention is a method for generating anti-4-1BB nanobodies or fusion proteins, (a) A step of culturing host cells according to the fifth aspect of the present invention under conditions suitable for generating anti-4-1BB nanobodies or fusion proteins, thereby obtaining a culture containing anti-4-1BB nanobodies or fusion proteins, (b) A step of isolating or recovering anti-4-1BB nanobodies or fusion proteins from the culture, A method including this is provided.

[0084] In the seventh aspect of the present invention, (a) an anti-4-1BB nanobody according to the first aspect of the present invention, or a fusion protein according to the second aspect of the present invention, (b) A coupling moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme, An immunoconjugate including this is provided.

[0085] In another preferred embodiment, the coupling portion is a drug or toxin.

[0086] In another preferred embodiment, the coupling portion is a detectable label.

[0087] In other preferred embodiments, the coupling portion is selected from fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, drug precursor activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or nanoparticles of any form.

[0088] In other preferred embodiments, the immunoconjugate comprises a polyvalent (e.g., divalent) form of the anti-4-1BB nanobody described in the first aspect of the present invention, or the fusion protein described in the second aspect of the present invention.

[0089] In other preferred embodiments, the term “multivalent” means that the immunoconjugate has an amino acid sequence comprising multiple repeats of the anti-4-1BB nanobody described in the first aspect of the present invention or the fusion protein described in the second aspect.

[0090] An eighth aspect of the present invention provides the use of an anti-4-1BB nanobody according to the first aspect of the present invention or a fusion protein according to the second aspect of the present invention in the manufacture of (a) a reagent for detecting 4-1BB molecules, or (b) a drug for treating tumors.

[0091] In other preferred embodiments, detection includes flow detection and cellular immunofluorescence detection.

[0092] In the ninth aspect of the present invention, (i) an anti-4-1BB nanobody according to the first aspect of the present invention, a fusion protein according to the second aspect of the present invention, or an immunoconjugate according to the seventh aspect of the present invention, (ii) A pharmaceutically acceptable carrier, A pharmaceutical composition containing the following is provided.

[0093] In another preferred embodiment, the pharmaceutical composition is in the form of an injectable preparation.

[0094] In another preferred embodiment, a drug for treating a tumor is prepared using a pharmaceutical composition, the tumor being selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, melanoma, head and neck cancer, nasopharyngeal cancer, thyroid tumor, tongue cancer, or a combination thereof.

[0095] In a tenth aspect of the present invention, one or more of the following uses of the anti-4-1BB nanobody described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention are provided. (i) Use for the detection of human 4-1BB molecules, (ii) Use for flow detection, (iii) Use for cellular immunofluorescence detection, (iv) Use for the treatment of tumors, and (v) Use for tumor diagnosis

[0096] In other preferred embodiments, the use is non-diagnostic and non-therapeutic.

[0097] In the eleventh aspect of the present invention, (i) A sequence of an anti-4-1BB nanobody described in the first aspect of the present invention or a fusion protein described in the second aspect of the present invention (ii) Tag sequences to aid expression and / or purification, as needed, Recombinant proteins containing the following are provided.

[0098] In other preferred embodiments, the tag array includes 6His tags, HA tags, Flag tags, Fc tags, or combinations thereof.

[0099] In another preferred embodiment, the recombinant protein specifically binds to the 4-1BB protein.

[0100] In a twelfth aspect of the present invention, an anti-4-1BB nanobody as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention is provided for use in preparing a drug, reagent, detection plate, or kit, The reagents, detection plates, or kits are used to detect 4-1BB protein in the sample. The drugs are used to treat or prevent various hematological malignancies and solid tumors.

[0101] In other preferred embodiments, the tumors include gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, adrenal tumor, melanoma, head and neck cancer, nasopharyngeal cancer, thyroid tumor, tongue cancer, or a combination thereof.

[0102] A thirteenth aspect of the present invention is a method for detecting 4-1BB protein in a sample, (1) A step of contacting a sample with an anti-4-1BB nanobody described in the first aspect of the present invention or a fusion protein described in the second aspect of the present invention, (2) A step to detect whether an antigen-antibody complex has been formed, Includes, The formation of the complex indicates the presence of the 4-1BB protein in the sample. A method is provided.

[0103] In other preferred embodiments, detection includes qualitative detection and quantitative detection.

[0104] A fourteenth aspect of the present invention is a method for treating a disease, A method is provided comprising the step of administering an anti-4-1BB nanobody described in the first aspect of the present invention, a fusion protein described in the second aspect of the present invention, or an immunoconjugate described in the seventh aspect of the present invention to a subject requiring such administration.

[0105] In other preferred embodiments, the subjects include mammals.

[0106] In another preferred embodiment, the mammal is a human.

[0107] In a fifteenth aspect of the present invention, a protein complex is provided which is formed by the interaction of a 4-1BB protein with an anti-4-1BB antibody or its antigen-binding fragment.

[0108] In other preferred embodiments, the interactions include hydrogen bonding and hydrophobic interactions.

[0109] In another preferred embodiment, the anti-4-1BB antibody is an anti-4-1BB nanobody.

[0110] In other preferred embodiments, the anti-4-1BB antibody is the anti-4-1BB nanobody described in the first aspect of the present invention.

[0111] In other preferred embodiments, the complex is formed by hydrogen bonding and / or hydrophobic interactions between the 4-1BB protein and the anti-4-1BB antibody or its antigen-binding fragment. The hydrogen bond comprises more than four (preferably more than five, more preferably more than six, most preferably seven) sites of action selected from the group consisting of Asp at position 118, Leu at position 123, Arg at position 130, Val at position 132, Cys at position 134, Gly at position 135, and Ser at position 137 within the 4-1BB protein. Furthermore, the hydrophobic interaction has an interface of action consisting of more than two (preferably more than three, more preferably more than four) sites selected from the group consisting of Leu at position 123, Val at position 124, Val at position 133, and Pro at position 136 within the 4-1BB protein. The amino acid numbering of the 4-1BB protein is based on the numbering of SEQ ID NO: 168.

[0112] In other preferred embodiments, the interface of hydrophobic interaction further comprises more than four (preferably more than five, more preferably more than six, most preferably more than seven or more than eight) sites selected from the group consisting of Val at position 32, Ala at position 33, Tyr at position 37, Leu at position 47, Ile at position 52, Tyr at position 97, Tyr at position 102, and Trp at position 115 within the anti-4-1BB antibody or its antigen-binding fragment. The amino acid numbering of the anti-4-1BB antibody or its antigen-binding fragment is based on the numbering of SEQ ID NO: 169 or 39.

[0113] In another preferred embodiment, the 4-1BB protein in the complex has the amino acid sequence of SEQ ID NO: 168.

[0114] In other preferred embodiments, the anti-4-1BB nanobody in the complex has the amino acid sequence of SEQ ID NO: 169 or 39.

[0115] In other preferred embodiments, the hydrogen bonds are located at Asp at position 118, Leu at position 123, Arg at position 130, Val at position 132, Cys at position 134, Gly at position 135, and / or Ser at position 137. The amino acid numbering is based on the numbering of SEQ ID NO: 168.

[0116] In other preferred embodiments, the hydrophobic interaction interfaces are located at Leu at position 123, Val at position 124, Val at position 133 and / or Pro at position 136 of the 4-1BB protein, and at Val at position 32, Ala at position 33, Tyr at position 37, Leu at position 47, Ile at position 52, Tyr at position 97, Tyr at position 102 and Trp at position 115 of the anti-4-1BB nanobody. The amino acid numbering of the 4-1BB protein is based on the numbering of SEQ ID NO: 168, and the amino acid numbering of the VHH chain of the anti-4-1BB nanobody is based on the numbering of SEQ ID NO: 169 or 39.

[0117] term To facilitate understanding of this disclosure, certain terms are defined first. In this application, unless otherwise specified herein, the following terms have the meanings set forth below. Other definitions are set out throughout this application.

[0118] As used herein, the term “about” may mean a value or composition within the tolerance range of a particular value or composition as determined by those skilled in the art, but this depends in part on how that value or composition is measured or determined.

[0119] In this specification, the terms “give” and “administer” are used interchangeably and mean physically introducing the product of the present invention into a target by any of the various methods and delivery systems known to those skilled in the art (e.g., intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes by injection or infusion).

[0120] Nanobody of the present invention In this specification, the terms “nanobody of the present invention,” “anti-4-1BB nanobody of the present invention,” and “4-1BB nanobody of the present invention” are used interchangeably and all refer to nanobody capable of specifically recognizing and binding to 4-1BB (including human 4-1BB). Particularly preferred are nanobody having a VHH chain having one amino acid sequence of any one of SEQ ID NOs: 1 to 40, and more preferably, nanobody having a VHH chain having one amino acid sequence of any one of SEQ ID NOs: 3, 35, 37, 38, 39, or 40.

[0121] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to a nanobody (VHH) composed of a single heavy chain variable region, constructed by cloning the antibody heavy chain variable region, and is the smallest antigen-binding fragment with full function. Typically, after obtaining an antibody that naturally lacks the constant region 1 (CH1) of the light and heavy chains, the heavy chain variable region of the antibody is cloned to construct a nanobody (VHH) composed of a single heavy chain variable region.

[0122] As used herein, the terms “antibody” or “immunoglobulin” refer to a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H), possessing identical structural characteristics. Each light chain is attached to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds differs between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also contains intrachain disulfide bonds arranged at regular intervals. Each heavy chain has a variable region (VH) at one end, followed by several constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other. The constant region of the light chain lies opposite the first constant region of the heavy chain, and the variable region of the light chain lies opposite the variable region of the heavy chain. Certain amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0123] As used herein, the term “variable” means that a portion of the antibody variable domain differs in sequence, contributing to the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the antibody variable domain. Variability is concentrated in three segments within the light and heavy chain variable domains, referred to as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portion of the variable domain is referred to as the framework region (FR). The natural heavy and light chain variable domains each contain four FR regions connected by three CDRs that form binding loops, which are roughly folded structures, and may sometimes form partially folded structures. The CDRs of each chain are adjacent by FR regions and, together with the CDRs of other chains, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant region does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as being involved in antibody-dependent cytotoxicity.

[0124] As is known to those skilled in the art, immunoconjugates and fusion expression products include conjugates obtained by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibody or fragment thereof of the present invention. The present invention further includes cell surface labels or antigens that can be bound to an anti-4-1BB protein antibody or fragment thereof.

[0125] In this specification, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0126] In this specification, the terms “variable region” and “complementarity-determining region (CDR)” are used interchangeably.

[0127] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions, namely CDR1, CDR2, and CDR3.

[0128] In a preferred embodiment of the present invention, the antibody heavy chain includes the heavy chain variable region and the heavy chain constant region described above.

[0129] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably and all mean polypeptides that specifically bind to the 4-1BB protein, such as proteins or peptides having a heavy chain variable region, which may or may not contain initiating methionine.

[0130] The present invention further provides additional proteins or fusion expression products comprising the antibody 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 variable region, provided that the heavy chain variable region is identical to, or at least 90% homologous to, the heavy chain of the antibody of the present invention, or at least 95% homologous.

[0131] Generally, the antigen-binding properties of an antibody can be explained by three specific regions located in the heavy chain variable region, called the variable region (CDR). This variable region is divided into four framework regions (FRs), but these four FRs have relatively conserved amino acid sequences and do not directly participate in the binding reaction. These CDRs form a cyclic structure, and the β-sheets formed by the FRs between them are in close proximity to each other in spatial structure. The CDRs of the heavy chain and the corresponding CDRs of the light chain constitute the antigen-binding site of the antibody. By comparing the amino acid sequences of antibodies of the same type, the amino acids constituting the FR or CDR region can be determined. The nanobody also has four framework regions (frameworks), of which the first, third, and fourth framework regions (framework region 1, framework region 3, and framework region 4) have high homology to human antibodies and do not need to be modified during the humanization process, but the second framework region, i.e., framework region 2 (framework 2), is preferably modified.

[0132] The variable region of the antibody heavy chain of the present invention is of particular interest because at least a portion of it is involved in binding to the antigen. Accordingly, the present invention includes molecules having an antibody heavy chain variable region having a CDR, insofar as the CDR has greater than 90% homology (preferably greater than 95%, most preferably greater than 98%) to the CDRs identified herein.

[0133] The present invention includes not only intact antibodies, but also antibody fragments having immunological activity, or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention further includes antibody fragments, derivatives, and analogs.

[0134] As used herein, the terms “fragment,” “derivative,” and “analog” mean polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. Polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides having one or more substituted conserved or non-conserved amino acid residues (preferably conserved amino acid residues) (such substituted amino acid residues may or may not be encoded by the genetic code), or (ii) polypeptides having substituents on 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, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence with a polypeptide sequence (e.g., a fusion protein formed by a leader sequence or secretion sequence or a sequence for purifying the polypeptide or a proprotein sequence, or by a 6His tag). In view of the disclosure herein, such fragments, derivatives, and analogs are within the scope of the art.

[0135] The antibody of the present invention refers to a polypeptide having 4-1BB protein-binding activity and containing the CDR region described above. This term further includes mutants of polypeptides having similar function to the antibody of the present invention and containing the CDR region described above. Such mutations include, but are not limited to, the deletion, insertion, and / or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and the addition of one or more (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids that have similar or similar properties usually does not change the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. This term also includes active fragments and active derivatives of the antibody of the present invention.

[0136] Polypeptide variants include homologous sequences, conserved variants, allelic variants, native variants, induced variants, proteins encoded by DNA capable of hybridizing with the DNA of the antibody of the present invention under high-stringent or low-stringent conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0137] The present invention further provides other polypeptides, such as fusion proteins, that include a single-domain antibody or a fragment thereof. In addition to substantially full-length polypeptides, the present invention further includes fragments of the single-domain antibody of the present invention. Typically, this 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.

[0138] In the present invention, "conservative variant of the antibody of the present invention" means a polypeptide formed by substituting up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids in the amino acid sequence with amino acids that have similar or close properties to those of the antibody of the present invention. Such conservative variant polypeptides are preferably produced by the amino acid substitutions listed in Table A. [Table 2]

[0139] The present invention further provides polynucleotide molecules encoding the aforementioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be either a coding strand or a non-coding strand.

[0140] The polynucleotides encoding mature polypeptides of the present invention include coding sequences that encode only mature polypeptides, coding sequences for mature polypeptides and various additional coding sequences, coding sequences for mature polypeptides (and any additional coding sequences) and non-coding sequences.

[0141] The term “polynucleotide encoding a polypeptide” may encompass polynucleotides that encode a polypeptide, or it may also encompass additional coding and / or non-coding sequences.

[0142] The present invention further relates to a polynucleotide that hybridizes to the above-described sequence and has at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to a polynucleotide that hybridizes to the polynucleotide of the present invention under stringent conditions. In the present invention, “stringent conditions” means (1) hybridization and elution at a lower ionic strength and a higher temperature, e.g., 0.2 × SSC, 0.1% SDS, 60°C, or (2) hybridization at 42°C, etc., in the presence of a denaturant, e.g., 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficol, etc., or (3) hybridization that occurs only when the identity between the two sequences exceeds at least 90%, preferably 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0143] The full-length nucleotide sequences of the antibodies or fragments thereof of the present invention can typically be obtained by PCR amplification, recombination, or artificial synthesis. A feasible method, particularly when the fragment length is short, is to synthesize the relevant sequence using artificial synthesis. Fragments with very long sequences are usually obtained by synthesizing multiple smaller fragments and then ligating them together. Furthermore, the heavy chain coding sequence can also be fused with an expression tag (e.g., 6His) to form a fusion protein.

[0144] Once a relevant sequence is obtained, it can be obtained in large quantities using recombination. Typically, the relevant sequence is cloned into a vector, transformed into cells, and then isolated from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules in their isolated forms.

[0145] Currently, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present invention can be obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) or cells known in the art. Furthermore, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0146] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or regulatory sequence. Such vectors can be used to transform appropriate host cells and express proteins.

[0147] 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. Typical examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium, fungal cells such as yeast, insect cells such as Drosophila S2 or Sf9, and animal cells such as CHO, COS7, and 293 cells.

[0148] Transformation of host cells using recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of taking up DNA can be collected after the exponential growth phase and treated with CaCl2 according to procedures well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation if necessary. When the host is a eukaryote, the following conventional mechanical DNA transfection methods can be used, namely calcium phosphate coprecipitation, microinjection, electroporation, and liposome packaging.

[0149] The resulting transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. The culture medium can be selected from a variety of conventional media depending on the host cells used. The cells are cultured under conditions suitable for host cell proliferation. After the host cells have proliferated to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0150] The recombinant polypeptides in the above-described method can be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant proteins can be isolated and purified by various separation methods utilizing their physical, chemical, and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional regeneration processes, treatment with protein precipitants (salting-out), centrifugation, osmotic disruption, superprocessing, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques or combinations thereof.

[0151] The antibodies of the present invention can be used alone or in combination with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified moieties, or any combination thereof.

[0152] Examples of detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0153] Examples of therapeutic agents that can be used in combination with the antibody of the present invention include, but are not limited to, 1. radionuclides, 2. biotoxins, 3. cytokines such as IL-2, 4. gold nanoparticles / nanorods, 5. viruses, 6. liposomes, 7. nanomagnetic particles, 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), 9. chemotherapeutic agents (e.g., cisplatin), or nanoparticles of any form.

[0154] The fusion protein of the present invention In this specification, "fusion protein of the present invention" means a fusion protein having both an anti-4-1BB nanobody and an immunoglobulin Fc fragment as described in the first aspect of the present invention.

[0155] In this invention, a fusion protein is provided, and the nanobody fusion protein has a structure represented by formula I from the N-terminus to the C-terminus. Z1-L-Z2 (Formula I) (In the formula, Z1 represents one or more VHH chains of the anti-4-1BB nanobody described in the first aspect of the present invention, Z2 represents the Fc fragment of immunoglobulin. (L represents any linker array)

[0156] Preferably, the immunoglobulin can be IgG1, IgG2, IgG3, or IgG4 (preferably IgG1, IgG2, or IgG4), or a different variant of IgG1.

[0157] In one embodiment, the fusion protein is a dimer, which is formed by disulfide bonds between the Fc fragments of Z2.

[0158] In one embodiment, L has an amino acid sequence selected from the group consisting of GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or combinations thereof.

[0159] In a preferred embodiment, Z2 has the amino acid sequence of SEQ ID NO: 167.

[0160] In other embodiments, Z2 has the same or substantially the same amino acid sequence as the amino acid sequence of SEQ ID NO: 167.

[0161] Preferably, the term “substantially identical” means that up to 50 amino acids differ (preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to 3), and that the differences include amino acid substitutions, deletions, or additions.

[0162] Preferably, the term “substantially identical” means that the amino acid sequences have at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to the corresponding amino acid sequence.

[0163] In a preferred embodiment of the present invention, the amino acid sequence of the fusion protein includes, from the N-terminus to the C-terminus, any sequence of SEQ ID NOs: 1 to 40 and the sequence of SEQ ID NO: 167. Any of the above-mentioned amino acid sequences may have 1 to 8 (preferably 1 to 5, more preferably 1 to 3) amino acid residues added, deleted, modified, and / or substituted as needed, further comprising a derived sequence that can maintain 4-1BB binding affinity.

[0164] The fusion protein of the present invention has the advantages of high dual-target binding affinity and strong specificity, thereby further enhancing antitumor immune function.

[0165] Pharmaceutical composition The present invention also provides compositions. Preferably, these compositions are pharmaceutical compositions comprising the antibodies or their active fragments or fusion proteins described above and a pharmaceutically acceptable carrier. Typically, such materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, the pH of which is usually about 5 to 8, preferably about 6 to 8, but the pH value can be changed depending on the properties of the formulated substance and the conditions to be treated. The prepared pharmaceutical compositions can be administered by conventional routes (e.g., intratumoral, intraperitoneal, intravenous, or topical administration).

[0166] The pharmaceutical composition of the present invention can be used directly to bind 4-1BB protein molecules and therefore can be used to treat tumors. Furthermore, additional therapeutic agents can be used in combination.

[0167] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-described single-domain antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, physiological saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation must be matched to the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injectable preparation by conventional methods, for example, using physiological saline or an aqueous solution containing glucose or other adjuvants. Pharmaceutical compositions such as injectable preparations and solutions are preferably manufactured under sterile conditions. The active ingredient is administered at a therapeutically effective dose, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. Furthermore, the polypeptide of the present invention may be used in combination with additional therapeutic agents.

[0168] When using pharmaceutical compositions, a safe and effective amount of immunoconjugate is administered to mammals. This safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases, less than or equal to about 50 mg / kg body weight, preferably between about 10 μg / kg body weight and about 10 mg / kg body weight. However, factors such as the route of administration and the patient's health condition should also be considered when determining a specific dosage, and this is within the scope of the skills of a skilled physician.

[0169] Labeled nanobody In preferred embodiments of the present invention, the nanobody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, gold colloid labels, colored labels, or fluorescent labels.

[0170] Gold colloid labeling can be carried out using methods known to those skilled in the art. In a preferred embodiment of the present invention, anti-4-1BB nanobodies are labeled with colloidal gold to obtain gold colloid-labeled nanobodies.

[0171] The anti-4-1BB nanobodies of the present invention possess good specificity and high titer.

[0172] Detection method The present invention also relates to a method for detecting the 4-1BB protein. This method generally comprises the following steps: obtaining a cell sample and / or a tissue sample; dissolving the sample in a medium; and detecting the level of the 4-1BB protein in the dissolved sample.

[0173] In the detection method of the present invention, the sample used is not particularly limited, but a typical example is a cell-containing sample present in a cell preservation solution.

[0174] kit The present invention also provides a kit comprising the antibody (or fragment thereof) or fusion protein or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, buffer solution, etc.

[0175] The present invention also provides a detection kit for detecting the level of 4-1BB, comprising an antibody capable of recognizing the 4-1BB protein, a lysis medium for dissolving the sample, and common reagents and buffers necessary for detection, such as various buffers, a detection label, and a detection substrate. This test kit can be used as an in vitro diagnostic device.

[0176] Purpose As described above, the nanobodies of the present invention have various biological and clinical application values, and their uses relate to the diagnosis and treatment of diseases related to 4-1BB, basic medical research, biological research, and other fields.

[0177] Preferred applications include clinical diagnosis and targeted therapy for 4-1BB. [Effects of the Invention]

[0178] Beneficial Effects of the Present Invention The present invention achieves one or more of the following technical effects. 1) The nanobodies of the present invention have a small molecular weight. 2) The nanobody of the present invention can bind to human 4-1BB protein with high specificity and simultaneously cross-bind to cynomolgus monkey protein. 3) The nanobodies of the present invention require external crosslinking to exhibit their activation function, and therefore possess excellent activity and safety. 4) The nanobodies of the present invention do not block the binding of 4-1BB ligand to 4-1BB in their natural state. 5) Urelumab, as an anti-4-1BB agonist molecule, has clinical hepatotoxicity due to its hyperactivating activity. An important advantage of the antibody of the present invention is that it has a different binding site to 4-1BB than urelumab and utomirumab, and its activating activity is between that of urelumab and utomirumab. [Brief explanation of the drawing]

[0179] [Figure 1A] Figure 1A shows the detection results of the first batch of the 4-1BB antibody of the present invention in binding to CHO cells that stably express human 4-1BB. [Figure 1B] Figure 1B shows the detection results of the second batch of the 4-1BB antibody of the present invention in binding to CHO cells that stably express human 4-1BB. [Figure 2A] Figure 2A shows the detection results of the first batch of the 4-1BB antibody of the present invention in binding to the cynomolgus monkey 4-1BB protein. [Figure 2B] Figure 2B shows the detection results of the second batch of the 4-1BB antibody of the present invention in binding to the cynomolgus monkey 4-1BB protein. [Figure 3A] Figure 3A shows the detection results of the first batch of the 4-1BB antibody of the present invention in the activation of primary T cells for IFN-γ generation. [Figure 3B] Figure 3B shows the detection results of the second batch of the 4-1BB antibody of the present invention in the activation of primary T cells for IFN-γ generation. [Figure 4A] Figure 4A shows the detection results of genetically modified offspring molecules of the L-Yr-13&14-16 molecule of the present invention in binding to CHO cells that stably express human 4-1BB. [Figure 4B] Figure 4B shows the detection results of genetically modified offspring molecules of the A-Na-19 molecule of the present invention in binding to CHO cells that stably express human 4-1BB. [Figure 5A] Figure 5A shows the detection results of genetically modified offspring molecules of the L-Yr-13&14-16 molecule of the present invention in binding to CHO cells that stably express cynomolgus monkey 4-1BB. [Figure 5B] Figure 5B shows the detection results of genetically modified offspring molecules of the A-Na-19 molecule of the present invention in binding to CHO cells that stably express cynomolgus monkey 4-1BB. [Figure 6A] Figure 6A shows the detection results of genetically modified offspring molecules of the L-Yr-13&14-16 molecule of the present invention in the activation of primary T cells for IFN-γ generation. [Figure 6B]Figure 6B shows the detection results of genetically modified offspring molecules of the A-Na-19 molecule of the present invention in the activation of primary T cells for IFN-γ generation. [Figure 7A] Figure 7A shows the detection results of genetically engineered offspring molecules of the L-Yr-13&14-16 molecule of the present invention in blocking the binding of the 4-1BB ligand to 4-1BB. [Figure 7B] Figure 7B shows the detection results of binding of the L-Yr-13&14-16-1 molecule of the present invention to the TNFRSF member. [Figure 8] Figure 8 shows a schematic diagram of the crystal of the 4-1BB-VHH composite of the present invention. [Figure 9] Figure 9 shows the crystal structure of the 4-1BB-VHH complex of the present invention. In this structure, green represents the 4-1BB protein, and blue represents the VHH chain of the anti-4-1BB nanobody. [Figure 10] Figure 10 shows the hydrogen bonding interaction interface in the crystal structure of the 4-1BB-VHH complex of the present invention. In this figure, green represents the 4-1BB protein, and blue represents the VHH chain of the anti-4-1BB nanobody. [Figure 11] Figure 11 shows the hydrophobic interaction interface in the crystal structure of the 4-1BB-VHH complex of the present invention. In this figure, green represents the 4-1BB protein, and blue represents the VHH chain of the anti-4-1BB nanobody. [Modes for carrying out the invention]

[0180] In this invention, the sequence numbers of the anti-4-1BB nanobodies and their CDRs involved are shown in Table B below. [Table 3]

[0181] Here, the CDR of each nanobody is defined by the IMGT numbering system. [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] Note: In Table C, sequence numbers 41-160 are CDR sequences. Some sequences may be identical, but for convenience of notation, different numbers are used.

[0182] A specific model for carrying out the present invention The present invention will be further described with the following specific examples. It should be understood that these examples are used solely for illustrative purposes and are not intended to limit the scope of the invention. Experimental methods in the following examples where specific conditions are not given are typically carried out under conventional conditions, such as those described in Sambrook et al., Molecular cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise specified, percentages (%) and parts are by weight. [Examples]

[0183] Example 1: Construction of a nanobody library 1.1 Immunization of animals Human 4-1BB antigen (purchased from AcroBiosystems) 1 mg was mixed with an equal volume of Freund's adjuvant, and two alpacas (llamas) were immunized with this mixture once a week for a total of four immunizations to stimulate B cells and express antigen-specific nanobodies. After the four immunizations, 50 mL of peripheral blood was collected from the alpacas, and lymphocytes were separated using lymphocyte isolation medium. Total RNA was extracted using the RNA extraction reagent Trizole (purchased from Invitrogen). Total cDNA of the alpacas was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0184] 1.2 Amplification of nanobody genes In the first round of PCR, the IgG2 and IgG3 sequences were amplified from the cDNA. [Table 12]

[0185] The PCR products from the first round were subjected to agarose gel electrophoresis, and a 750 bp fragment recovered by cutting the gel was used for VHH sequence amplification in the second round. The primers for the second round of PCR amplification were as follows: [Table 13]

[0186] The PCR product from the second round was used as a template to perform the third round of PCR, and homology arms were added to the VHH gene. The primers for the third round of PCR amplification were as follows: [Table 14]

[0187] Target fragments were recovered using a PCR purification kit (purchased from QIAGEN).

[0188] 1.3 Library Construction A linearized yeast display vector and the third round PCR product were mixed and electrotransformed into Saccharomyces cerevisiae (ATCC® 20828) to construct an anti-4-1BB nanobody library from two alpacas.

[0189] Example 2: Screening of 4-1BB nanobodies 2.1 Biotin labeling of human 4-1BB protein Human 4-1BB protein (purchased from AcroBiosystems) was dissolved in an appropriate amount of twice-distilled water, and biotin was dissolved according to the instructions for the biotin labeling kit (purchased from Thermo). The mixture was then mixed with the protein solution and incubated at 4°C for 2 hours. Excess biotin was removed using a desalting column (purchased from Thermo). All desalting column pretreatment and sample recovery procedures followed the instructions in the product manual.

[0190] 2.2 MACS (Magnetic Activated Cell Sorting) Concentration of Yeast Specifically Capable of Binding to 4-1BB The VHH library constructed in Example 1 was seeded in SD-CAA growth medium (1 L of SD-CAA growth medium containing 6.7 g of YNB, 5 g of casamino acid, 13.62 g of Na2HPO4·12H2O, 7.44 g of NaH2PO4, and 2% glucose) and cultured overnight at 30°C and 225 rpm. An appropriate amount of yeast cells was collected, centrifuged at 3000 rpm for 5 minutes to remove the medium, and the yeast cells were resuspended in SD-CAA induction medium and induced overnight. The library concentration after induction was measured, an appropriate amount of yeast cells was collected, and the medium was removed by centrifugation. The yeast cells were resuspended in 50 mL of washing buffer (PBS + 0.5% BSA + 2 mM EDTA), centrifuged, and the supernatant was removed. The yeast cells were resuspended in 10 mL of washing buffer.

[0191] Biotin-labeled 4-1BB protein (final concentration 100 mM) was added, incubated at room temperature for 30 minutes, and yeast cells were collected by centrifugation. The yeast cells were washed three times with 50 mL of washing buffer. The yeast cells were resuspended in 5 mL of washing buffer, 200 μL of SA magnetic beads (purchased from Miltenyi) were added, and the column was inverted and incubated for 10 minutes. The mixture of yeast cells and magnetic beads was washed three times with washing buffer and the mixture was added to an LS column (purchased from Miltenyi). The LS column was placed on a magnetic stand and washed with washing buffer to remove nonspecifically bound yeast cells. The column was removed from the magnetic stand, and washing buffer was added to elute the yeast cells. The eluted yeast cells were centrifuged and transferred to 200 mL of SD-CAA growth medium for amplification.

[0192] 2.3 Flow cytometry sorting to obtain high-affinity yeast cells MACS-enriched yeast cells were seeded in SD-CAA growth medium and cultured overnight in a shaking flask at 30°C and 225 rpm. The yeast cells were resuspended in SD-CAA induction medium (1 L of SD-CAA induction medium containing 6.7 g of YNB, 5 g of casamino acid, 13.62 g of Na2HPO4·12H2O, 7.44 g of NaH2PO4, along with 2% galactose, 2% raffinose, and 0.1% glucose) and induced overnight. A 1:200 dilution of anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotin-labeled 4-1BB antigen were added, and the cells were incubated at room temperature for 10 minutes. Yeast cells were washed three times with PBS, and then 1:500 dilution of goat anti-mouse IgG(H+L) Alexa Fluor Plus 488 (purchased from Invitrogen) and streptavidin APC-conjugated fluorescent antibody (purchased from Invitrogen) were added. The cells were incubated in the dark at 4°C for 15 minutes. The cells were resuspended with 2 mL of PBS, and yeast cells with high binding ability to the 4-1BB antigen were sorted using a BD FACS Aria II instrument.

[0193] 2.4 Gene acquisition of candidate molecules for 4-1BB nanobody Yeast cells with high binding affinity to the 4-1BB antigen, obtained by MACS and FACS enrichment, were cultured overnight in SD-CAA amplification medium at 30°C and 225 rpm. Plasmids were extracted using a yeast plasmid extraction kit (purchased from Tiangen). These plasmids were electrotransformed into Top 10 competent cells (purchased from Tiangen), coated onto ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were collected and sequenced to obtain the VHH gene sequence.

[0194] Example 3: Construction and expression / purification of heavy chain antibody fusion protein 3.1 Construction of antibody genes into pCDNA3.1 expression vectors The VHH gene sequence obtained in Example 2 was ligated to the Fc fragment of human IgG1 (LALA mutation), and the linearized pCDNA3.1 vector was double-digested with homologous recombinase (purchased from Vazyme) and EcoRI / NotI, following the procedure as described in the product instructions. The homologous recombination product transformed Top10 competent cells, coated on ampicillin-resistant plates, cultured overnight at 37°C, and single clones were collected and sequenced.

[0195] 3.2 Cell transfection Plasmids were introduced into Expi-CHO cells using the ExpiCHO® Expression System Kit (purchased from Thermo). Transfection was performed according to the product instructions. After culturing the cells for 5 days, the supernatant was collected, and the target protein was purified by sorting using Protein A magnetic beads (purchased from Nanjing Jinsirui Biotechnology Co., Ltd.). The magnetic beads were resuspended in an appropriate amount of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1 to 4 times the volume of the magnetic beads), added to the sample to be purified, and incubated at room temperature for 1 hour with gentle shaking. The sample was placed on a magnetic stand (purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. Elution buffer (0.1M sodium citrate, pH 3.2) was added in an amount 3 to 5 times the volume of the magnetic beads, and after shaking at room temperature for 5 to 10 minutes, the mixture was returned to the magnetic stand, the elution buffer was collected, and the mixture was transferred to a collection tube to which neutralization buffer (1M Tris, pH 8.54) was added and thoroughly mixed. The purified 4-1BB antibody fusion protein sample was used in Examples 4 to 7 below.

[0196] Example 4: Affinity measurement of 4-1BB antibody fusion protein ForteBio affinity measurement was performed according to an existing method (Estep, P et al., Solution-based measurement of high-throughput antibody-antigen affinity and epitope classification, MAbs, 2013.5(2):p.270-8). Specifically, the sensor was equilibrated offline in assay buffer for 30 minutes, then detected online for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. Next, the sensor was treated with 100 nM 4-1 BB antigen for 5 minutes, then transferred to PBS and dissociated for 5 minutes. Dynamic analysis was performed using a 1:1 binding model.

[0197] The experimental results are shown in Table 4 below. [Table 15]

[0198] These results indicate that the monovalent affinity of the anti-4-1BB molecule of the present invention is at the 1E-08 level, thus meeting the requirements for the development of an agonist molecule.

[0199] Example 5: Binding of 4-1BB antibody fusion protein to human 4-1BB CHO-S cells CHO cells overexpressing human 4-1BB (CHO-h4-1BB cells) were generated by transfection with the pCHO1.0 vector (purchased from Invitrogen) encoding human 4-1BB cDNA cloned into the MCS (multiple cloning site). The cell density of the proliferated CHO-h4-1BB cells was 2 × 10⁻⁶. 6 The cells were adjusted to cells / mL and added to 96-well flow plates at 100 μL / well. The plates were then centrifuged and prepared for subsequent use. The purified 4-1BB antibody was diluted 3-fold in PBS (starting at 400 nM, for a total of 12 samples). The diluted samples were added to the aforementioned 96-well flow plates containing cells at 100 μL / well, incubated at 4°C for 30 minutes, and washed twice with PBS. Goat F(ab')2 anti-human IgG-Fc(PE) (purchased from Abcam) diluted 100-fold in PBS was added at 100 μL / well, incubated at 4°C for 30 minutes, and washed twice with PBS. Cells resuspended in PBS were added at 100 μL / well, detected using a CytoFlex (Bechman) flow cytometer, and the corresponding MFI was calculated.

[0200] The experimental results are shown in Figures 1A and 1B.

[0201] These results show that all purified samples of the present invention have binding activity to CHO-h4-1BB cells, and the activity of several purified samples is equivalent to that of the control antibody urelumab (U.S. Patent Application Publication No. 20090068192).

[0202] Example 6: Binding of 4-1BB antibody fusion protein to cynomolgus monkey 4-1BB protein Cynomolgus monkey 4-1BB-his protein (purchased from ACRO) was dissolved according to the instructions, diluted to 1 μg / mL with ELISA coating solution (purchased from Shanghai Sangon), coated ELISA plates with 100 μL / well, left overnight at 4°C, washed three times with PBST, and blocked with 5% BSA (purchased from Shanghai Sangon) at room temperature for 1 hour. The coating solution was discarded, and 100 μL / well of 4-1BB antibody, serially diluted 3-fold with 1% BSA (starting at 200 nM and totaling 12 dilutions), was added and incubated at room temperature for 2 hours. After washing three times with PBST, 100 μL / well of goat anti-human IgG-Fc-HRP (purchased from abcam), diluted with 1% BSA, was added and incubated at room temperature for 1 hour. The samples were washed three times with PBST, 100 μL / well of ELISA colorimetric solution (purchased from Solarbio) was added, the mixture was allowed to react at room temperature for 3 minutes, 50 μL / well of ELISA stop solution (purchased from Solarbio) was added, and the absorbance value at 450 nm was read.

[0203] The experimental results are shown in Figures 2A and 2B. Several purified samples of the present invention (e.g., L-Yr-13&14-16 and A-Na-19) and the control antibody utomirumab showed good binding activity to the cynomolgus monkey 4-1BB protein, while the control antibody urelumab (US Patent Application Publication No. 20090068192) showed weak binding activity to the cynomolgus monkey 4-1BB protein.

[0204] Example 7: Measurement of primary T cell activation by 4-1BB antibody fusion protein OKT-3 antibody (purchased from Biolegend) was diluted to 1 μg / mL in sterile PBS (purchased from Hyclone), coated with 50 μL / well of this solution onto 96-well cell culture flat-bottom plates (purchased from Thermo), and simultaneously added 50 μL / well of anti-4-1BB antibody serially diluted in PBS. The cells were incubated at 37°C for 2 hours. Frozen human PBMCs (purchased from Shanghai Saili) were revived, and T cells were isolated using a human T cell isolation and purification kit (purchased from Stemcell). The T cells were resuspended in X-VIVO15 medium (purchased from LONZA) and the cell density was 0.5 × 10⁶. 6 The solution was adjusted to a concentration of cells / mL and prepared for subsequent use. After antibody coating was complete, the coating solution was discarded, the cells were washed twice with PBS, the PBS was discarded, and 200 μL / well of the aforementioned T cell suspension was added. The cells were incubated at 37°C and 5% CO2 for 5 days, and the supernatant was collected to detect the IFN-γ content.

[0205] The experimental results are shown in Figures 3A and 3B. Several purified samples of the present invention were able to activate human primary T cells and secrete IFN-γ, some samples (e.g., A-Na-16, A-Na-18, A-Na-19) had activity comparable to the control antibody urelumab (US Patent Application Publication No. 20090068192), and some samples (e.g., L-Yr-13&14-16, L-Yr-13&14-17) had activity between urelumab and utomirumab.

[0206] Example 8: Humanization and expression of 4-1BB antibody To reduce the immunogenicity of monoclonal antibodies in humans, L-Yr-13&14-16 antibodies and A-Na-19 antibodies were humanized. Humanization was performed using the general framework transplantation method for VHH humanization, and simultaneously, some amino acids of antibody framework 2 (framework 2) were mutated according to a method reported in the literature (Vincke, C., et al., General strategy for humanizing camelid single-domain antibodies and identifying universal humanized nanobody scaffolds. J Biol Chem 284 (5): 3273-3284). Sequences of sequence numbers 39-40 and 37-38 were obtained, respectively.

[0207] In this study, the level of humanization of the humanized sequences of L-Yr-13&14-16 antibodies and A-Na-19 antibodies was evaluated using IMGT (http: / / www.imgt.org), and the results are shown in Table 5.

[0208] [Table 16]

[0209] The protein construction and expression purification methods were the same as in Example 3. The purified genetically modified antibody was tested for binding to human 4-1BB CHO-S cells, and the detection method was the same as in Example 4. The results are shown in Figures 4A and 4B. The genetically modified antibody maintained its binding activity to human 4-1BB CHO-S cells.

[0210] The binding of the purified genetically engineered antibody to cynomolgus 4-1BB CHO-S cells was tested. The results are shown in FIGS. 5A and 5B. From these results, it was found that the genetically engineered HZ-L-Yr-13&14-16 antibody maintained the binding activity to cynomolgus 4-1BB CHO-S cells, while the genetically engineered A-Na-19 antibodies HZ-A-Na-19-1 and HZ-A-Na-19-2 showed a significant decrease in the binding activity to cynomolgus 4-1BB CHO-S cells.

[0211] The purified genetically engineered antibody was tested for the activation of primary T cells, and the detection method was the same as in Example 7. The results are shown in FIGS. 6A and 6B. From these results, it was found that the genetically engineered HZ-L-Yr-13&14-16-1 and HZ-A-Na-19-1 maintained the activity to activate primary T cells.

[0212] Example 9: 4-1BB Ligand Blocking Test The grown CHO-h4-1BB cells were adjusted to a cell density of 2×10 6 cells / mL, added to a 96-well flow plate at 100 μL / well, and centrifuged in preparation for subsequent use. The purified 4-1BB antibody was diluted to 400 nM with PBS, and the above diluted sample was added to the above cell-containing 96-well flow plate at 50 μL / well and incubated at 4° C. for 30 minutes. Biotin-4-1BB ligand protein (purchased from ACRO) diluted to 1 μg / mL with PBS was added at 50 μL / well and incubated at 4° C. for 30 minutes. After washing twice with PBS, SAPE (purchased from Thermo) diluted 200-fold with PBS was added at 100 μL / well, incubated at 4° C. for 30 minutes, and washed twice with PBS. PBS was added at 100 μL / well to resuspend the cells, and detection was performed with a CytoFlex (manufactured by Bechman) flow cytometer, and the corresponding MFI was calculated.

[0213] The results are shown in Fig. 7A. From these results, it was found that the genetically engineered 4-1BB antibody HZ-L-Yr-13&14-16-1 has no ligand blocking activity, the control antibody utomilumab (US Patent Application Publication No. 20120237498) has ligand blocking activity, and uremlumab has partial blocking activity.

[0214] Example 10: Detection of antibody-TNFRSF member binding Human 4-1BB-his, GITR-his, OX40-his, CD40-his proteins (purchased from ACRO) were dissolved according to the instructions, diluted to 1 μg / mL with ELISA coating solution (purchased from Shanghai Sangon), and ELISA plates were coated at 100 μL / well and left overnight at 4°C. After washing three times with PBST, 5% BSA (purchased from Shanghai Sangon) was added at 200 μL / well and blocked at room temperature for 1 hour. The coating solution was discarded, and the 4-1BB antibody diluted with 1% BSA was added to three replicate wells at 100 μL / well and 200 nM and incubated at room temperature for 2 hours. After washing three times with PBST, goat anti-human IgG-Fc-HRP (purchased from abcam) diluted with 1% BSA was added at 100 μL / well and incubated at room temperature for 1 hour. After washing three times with PBST, ELISA chromogenic solution (purchased from Solarbio) was added at 100 μL / well and reacted at room temperature for 3 minutes, and ELISA stop solution (purchased from Solarbio) was added at 50 μL / well, and the absorbance value at 450 nm was read.

[0215] The results are shown in Fig. 7B. From these results, it was found that the HZ-L-Yr-13&14-16-1 antibody binds only to the 4-1BB protein and does not bind to other members of the tumor necrosis factor receptor superfamily. The antibody of the present invention has good specificity and was shown to not activate other members of the tumor necrosis factor receptor superfamily in vivo, thereby avoiding potential side effects.

[0216] Example 11: Identification of the crystal structure of the 4-1BB and VHH segment complex. To further investigate the binding mode of the 4-1BB nanobody of the present invention to its antigen protein, in this example, L-Yr-13&14-16-1 was selected and an antigen complex crystallization experiment was performed.

[0217] Here, in order to promote crystal formation, the amino acid sequence of the anti-4-1BB antibody in the final crystal complex, after optimization, was set to the sequence (SEQ ID NO: 169) formed by adding Leu and Gly to the C-terminus of the VHH chain sequence of L-Yr-13&14-16-1 (i.e., SEQ ID NO: 39). It should be noted that the Leu and Gly residues added at the C-terminus are merely a predetermined technical operation to promote crystallization and do not affect the binding mode of the nanobody L-Yr-13&14-16-1 to the human 4-1BB protein.

[0218] In this experiment, the crystal structure of the 4-1BB-VHH segment complex was identified using X-ray diffraction. Both human 4-1BB protein (SEQ ID NO: 168) and anti-4-1BBVHH protein (SEQ ID NO: 169) were expressed using the HEK293 system. A complex sample for crystallization was prepared by mixing 4-1BB with VHH in a 1:1 molar ratio. The complex (8.5 mg / mL) was mixed with a crystallization reagent in a 1:1 ratio and crystallized at 18°C. The crystals were observed after 4 days under the culture conditions of the JBK kit, and their crystalline morphology is shown in Figure 8.

[0219] A single crystal was selected for X-ray diffraction experiments using the Shanghai light source, and diffraction data with a resolution of 3.14 Å was obtained. Data processing was performed using XDS software. Using molecular substitution, the crystal phases were identified using the structures of 4-1BB (PDB ID: 6mgp) and VHH (PDB ID: 4xt1) as models. The crystal structure was refined using Refmac5. Model inspection, manual reconstruction, and structural verification were performed using COOT. The composite crystal belonged to the P41212 space group, and its unit cell parameters were as follows: a=106.85 Å, b=106.85 Å, c=146.41 Å, α=90.00°, β=90.00°, γ=90.00°. Statistical values ​​for specific crystal data are shown in Table 6. [Table 17]

[0220] The crystal structure of the 4-1BB-VHH complex obtained after structural analysis is shown in Figure 9. Epitope analysis revealed that the main hydrogen bonds between 4-1BB and VHH are concentrated in amino acids such as Asp118, Leu123, Arg130, Val132, Cys134, Gly135, and Ser137 of 4-1BB (Figure 10). Furthermore, Leu123, Val124, Val133, and Pro136 of 4-1BB and Val32, Ala33, Tyr37, Leu47, Ile52, Tyr97, Tyr102, and Trp115 of VHH constitute a hydrophobic interaction interface (Figure 11).

[0221] The binding site for urelumab to 4-1BB is located at the N-terminus of 4-1BB, and the amino acids of 4-1BB mainly involved in binding were Pro27, Asn40, Asn42, and Gln43. Utomirumab mainly binds to CRD3 (cysteine-rich domain) and CRD4 of 4-1BB, and the amino acids of 4-1BB mainly involved in binding were Arg66, Gly96, Ser100, Cys102, Lys114, Arg130, and Arg134 (Chin SM, Kimberlin CR, Roe-Zurz Z, et al. Structure of the 4-1BB / 4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab. Nat Commun 2018;9:4679.). Furthermore, theoretically, since the binding epitope of utomirumab was in a competitive relationship with the 4-1BB ligand in space, it is thought to have a certain blocking effect on the binding of 4-1BB to that ligand.

[0222] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will be able to make various changes and substitutions to the details based on all the disclosed teachings, and it will be understood that all such changes are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Sequence Listing Free-Text

[0223] SEQ ID NO: 1: Single domain antibody SEQ ID NO: 2: Single domain antibody SEQ ID NO: 3: Single domain antibody SEQ ID NO: 4: Single domain antibody SEQ ID NO: 5: Single domain antibody SEQ ID NO: 6: Single domain antibody SEQ ID NO: 7: Single domain antibody SEQ ID NO: 8: Single domain antibody SEQ ID NO: 9: Single domain antibody SEQ ID NO: 10: Single domain antibody SEQ ID NO: 11: Single domain antibody SEQ ID NO: 12: Single domain antibody SEQ ID NO: 13: Single domain antibody SEQ ID NO: 14: Single domain antibody SEQ ID NO: 15: Single domain antibody SEQ ID NO: 16: Single domain antibody SEQ ID NO: 17: Single domain antibody SEQ ID NO: 18: Single domain antibody SEQ ID NO: 19: Single domain antibody SEQ ID NO: 20: Single domain antibody SEQ ID NO: 21: Single domain antibody SEQ ID NO: 22: Single domain antibody SEQ ID NO: 23: Single domain antibody SEQ ID NO: 24: Single domain antibody SEQ ID NO: 25: Single domain antibody SEQ ID NO: 26: Single domain antibody SEQ ID NO: 27: Single domain antibody Sequence ID No. 28: Single-domain antibody Sequence ID No. 29: Single-domain antibody Sequence ID No. 30: Single-domain antibody Sequence ID 31: Single-domain antibody Sequence ID No. 32: Single-domain antibody Sequence ID 33: Single-domain antibody Sequence ID No. 34: Single-domain antibody Sequence ID No. 35: Single-domain antibody Sequence ID No. 36: Single-domain antibody Sequence ID No. 37: Single-domain antibody Sequence ID No. 38: Single-domain antibody Sequence ID 39: Single-domain antibody Sequence ID No. 40: Single-domain antibody Sequence ID 41: CDR Sequence ID 42: CDR Sequence ID 43: CDR Sequence ID 44: CDR Sequence ID 45: CDR Sequence ID 46: CDR Sequence ID 47: CDR Sequence ID 48: CDR Sequence ID 49: CDR Sequence ID 50: CDR Sequence ID 51: CDR Sequence ID 52: CDR Sequence ID 53: CDR Sequence ID 54: CDR Sequence ID 55: CDR Sequence ID 56: CDR Sequence ID 57: CDR Sequence ID 58: CDR Sequence ID 59: CDR Sequence ID 60: CDR Sequence ID 61: CDR Sequence ID 62: CDR Sequence ID 63: CDR Sequence ID 64: CDR Sequence ID 65: CDR Sequence ID 66: CDR Sequence ID 67: CDR Sequence ID 68: CDR Sequence ID 69: CDR Sequence ID 70: CDR Sequence ID 71: CDR Sequence ID 72: CDR Sequence ID 73: CDR Sequence ID 74: CDR Sequence ID 75: CDR Sequence ID 76: CDR Sequence ID 77: CDR Sequence ID 78: CDR Sequence ID 79: CDR Sequence ID 80: CDR Sequence ID 81: CDR Sequence ID 82: CDR Sequence ID 83: CDR Sequence ID 84: CDR Sequence ID 85: CDR Sequence ID 86: CDR Sequence ID 87: CDR Sequence ID 88: CDR Sequence ID 89: CDR Sequence ID 90: CDR Sequence ID 91: CDR Sequence ID 92: CDR Sequence ID 93: CDR Sequence ID 94: CDR Sequence ID 95: CDR Sequence ID 96: CDR Sequence ID 97: CDR Sequence ID 98: CDR Sequence ID 99: CDR Sequence ID 100: CDR Sequence ID 101: CDR Sequence ID 102: CDR Sequence ID 103: CDR Sequence ID 104: CDR Sequence ID 105: CDR Sequence ID 106: CDR Sequence ID 107: CDR Sequence ID 108: CDR Sequence ID 109: CDR Sequence ID 110: CDR Sequence ID 111: CDR Sequence ID 112: CDR Sequence ID 113: CDR Sequence ID 114: CDR Sequence ID 115: CDR Sequence ID 116: CDR Sequence ID 117: CDR Sequence ID 118: CDR Sequence ID 119: CDR Sequence ID 120: CDR Sequence ID 121: CDR Sequence ID 122: CDR Sequence ID 123: CDR Sequence ID 124: CDR Sequence ID 125: CDR Sequence ID 126: CDR Sequence ID 127: CDR Sequence ID 128: CDR Sequence ID 129: CDR Sequence ID 130: CDR Sequence ID 131: CDR Sequence ID 132: CDR Sequence ID 133: CDR Sequence ID 134: CDR Sequence ID 135: CDR Sequence ID 136: CDR Sequence ID 137: CDR Sequence ID 138: CDR Sequence ID 139: CDR Sequence ID 140: CDR Sequence ID 141: CDR Sequence ID 142: CDR Sequence ID 143: CDR Sequence ID 144: CDR Sequence ID 145: CDR Sequence ID 146: CDR Sequence ID 147: CDR Sequence ID 148: CDR Sequence ID 149: CDR Sequence ID 150: CDR Sequence ID 151: CDR Sequence ID 152: CDR Sequence ID 153: CDR Sequence ID 154: CDR Sequence ID 155: CDR Sequence ID 156: CDR Sequence ID 157: CDR Sequence ID 158: CDR Sequence ID 159: CDR Sequence ID 160: CDR Sequence ID 161: Primer Sequence ID 162: Primer Sequence ID 163: Primer Sequence ID 164: Primer Sequence ID 165: Primer Sequence ID 166: Emulator Sequence ID 167: Fc fragment of human IgG1 Sequence ID 168: Human 4-1BB protein SEQ ID NO: 169: Amino acid sequence of anti-4-1BB antibody in crystal complex Sequence ID 170: Framework 1 Sequence ID 171: Framework 2 Sequence ID 172: Framework 2 Sequence ID 173: Framework 3 Sequence ID 174: Framework 4

Claims

1. An anti-4-1BB single-domain antibody containing a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 to CDR3, CDR1 having the amino acid sequence of SEQ ID NO: 79, CDR2 having the amino acid sequence of SEQ ID NO: 119, and CDR3 having the amino acid sequence of SEQ ID NO:

159.

2. The anti-4-1BB single-domain antibody according to claim 1, comprising framework regions 1 to 4, wherein any one, two, three, or all four of the framework regions 1 to 4 are humanized.

3. The anti-4-1BB single-domain antibody according to claim 2, wherein the framework region 1 has the amino acid sequence of SEQ ID NO: 170, the framework region 2 has the amino acid sequence of SEQ ID NO: 171 or SEQ ID NO: 172, the framework region 3 has the amino acid sequence of SEQ ID NO: 173, and the framework region 4 has the amino acid sequence of SEQ ID NO:

174.

4. Satisfying at least one of the following conditions (1) to (4): (1) K against 4-1BB antigen Ｄ is less than E-07, less than 5E-08, less than 4E-08, less than 3E-08, or less than 2E-08. (2) The binding site for 4-1BB antigen is different from that of urelumab and utomirumab. (3) It specifically binds to human 4-1BB and simultaneously cross-binds to cynomolgus monkey 4-1BB. (4) It does not block the binding of the 4-1BB ligand to 4-1BB in its natural state. The anti-4-1BB single-domain antibody according to any one of claims 1 to 3.

5. An anti-4-1BB single-domain antibody according to any one of claims 1 to 4, having one amino acid sequence of any one of sequence numbers 39, 35, or 40.

6. A fusion protein comprising an anti-4-1BB single-domain antibody according to any one of claims 1 to 5 and an Fc fragment of human IgG or a constant region of human IgG.

7. The fusion protein according to claim 6, wherein, according to the EU numbering system, the Fc fragment of human IgG or the heavy chain constant region of human IgG comprises the L234A mutation and the L235A mutation, or the Fc fragment of human IgG further comprises the G237A mutation.

8. The Fc fragment of human IgG is either a human IgG1 Fc fragment or The heavy chain steady region of human IgG is the heavy chain steady region of human IgG1. The fusion protein according to claim 6 or claim 7.

9. The fusion protein according to any one of claims 6 to 8, wherein the human IgG1 Fc fragment has the amino acid sequence of SEQ ID NO:

167.

10. The fusion protein according to any one of claims 6 to 9, wherein the Fc fragment of human IgG or the constant region of human IgG is directly or via a linker fragment ligated to the C-terminus of the anti-4-1BB single-domain antibody.

11. An isolated nucleic acid molecule encoding an anti-4-1BB single-domain antibody according to any one of claims 1 to 5 or a fusion protein according to any one of claims 6 to 10.

12. A vector comprising an isolated nucleic acid molecule as described in claim 11.

13. A host cell containing the isolated nucleic acid molecule described in claim 11 or the vector described in claim 12.

14. A method for preparing an anti-4-1BB single-domain antibody according to any one of claims 1 to 5 or a fusion protein according to any one of claims 6 to 10, comprising the steps of: culturing a host cell according to claim 13 under appropriate conditions; and recovering the anti-4-1BB single-domain antibody or fusion protein from the cell culture.

15. A conjugate comprising an antibody portion and a coupling portion, wherein the antibody portion is an anti-4-1BB single-domain antibody according to any one of claims 1 to 5 or a fusion protein according to any one of claims 6 to 10, and the coupling portion is a detectable label.

16. The conjugate according to claim 15, wherein the coupling portion is a radioactive isotope, a fluorescent substance, a light-emitting substance, a coloring substance, or an enzyme.

17. A kit comprising an anti-4-1BB single-domain antibody according to any one of claims 1 to 5 or a fusion protein according to any one of claims 6 to 10, or a conjugate according to claim 15 or claim 16.

18. Use of an anti-4-1BB single-domain antibody according to any one of claims 1 to 5 or a fusion protein according to any one of claims 6 to 10 in the preparation of a kit for detecting the presence or level of 4-1BB in a sample.

19. A pharmaceutical composition comprising an anti-4-1BB single-domain antibody according to any one of claims 1 to 5, a fusion protein according to any one of claims 6 to 10, or a conjugate according to claim 15 or claim 16, and a pharmaceutically acceptable excipient.

20. Use of an anti-4-1BB single-domain antibody according to any one of claims 1 to 5, a fusion protein according to any one of claims 6 to 10, or a conjugate according to claim 15 or claim 16 in the manufacture of a drug for the prevention and / or treatment of malignant tumors or autoimmune diseases.

21. The use according to claim 20, wherein the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, stomach cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer.

22. The use according to claim 20, wherein the autoimmune disease is selected from the group consisting of autoimmune encephalomyelitis, loop-like syndrome, and collagen-induced arthritis.

23. An anti-4-1BB single-domain antibody according to any one of claims 1 to 5, used for the treatment and / or prevention of malignant tumors or autoimmune diseases.

24. The anti-4-1BB single-domain antibody according to claim 23, wherein the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer.

25. The anti-4-1BB single-domain antibody according to claim 23, wherein the autoimmune disease is selected from the group consisting of autoimmune encephalomyelitis, loop-like syndrome, and collagen-induced arthritis.

26. A fusion protein according to any one of claims 6 to 10, used for the treatment and / or prevention of malignant tumors or autoimmune diseases.

27. The fusion protein according to claim 26, wherein the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, breast cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer.

28. The fusion protein according to claim 26, wherein the autoimmune disease is selected from the group consisting of autoimmune encephalomyelitis, loop-like syndrome, and collagen-induced arthritis.

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